Methods and compositions for improving fertility

Abstract

N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA (mRNA). The present invention relates to a writing protein complex that is installed by a writing protein complex and can be reversed by erasure proteins such as fat mass and obesity-related proteins (FTO). Although wide studies have been made, the main physiological substrate of FTO in the tissue and development of mammals is still unclear. The aspects disclosed by the invention show that FTO mediates m6A demethylation of emera-1 LINE1 RNA in a mouse embryonic stem cell (mESC), the abundance and local chromatin state of the LINE1 RNA are regulated and controlled, and further the transcription of the LINE1-containing gene is regulated. The m6A demethylation of the FTO-mediated LINE1 RNA also plays a role in regulating and controlling the chromatin state and gene expression in the development process of mouse oocytes and embryos. FTO overexpression to modulate fertility is also disclosed.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 338,332, filed May 4, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the fields of biology, medicine, veterinary medicine and molecular biology. Background Art

[0003] N 6 -methyladenosine (m 6 A) is the most common internal modification of mammalian mRNA, which is regulated by writer proteins and eraser proteins, which affect the fate of transcripts through reader proteins (1-3). Fat mass and obesity-associated protein (FTO) was the first mRNA to be shown to remove mRNA. 6 A RNA demethylase (4). FTO is known to be involved in mammalian development and human diseases; for example, Fto - / - Mice exhibit severe developmental defects (5, 6). Extensive functional characterization in human cancer cells has shown that FTO localizes to the cytoplasm of cells and removes mRNA from mRNA transcripts, which contributes to cancer progression. 6 A(7-12); however, similar activity is not evident in mouse and human tissues, where FTO tends to exhibit nuclear localization (13). 6 Another form of A, m 6 A m , which is enriched in the caps of some mRNAs and certain small nuclear RNAs (snRNAs), has also been identified as a substrate for FTO (14-16). 6 A m The methyltransferase PCIF1 caused only mild cellular effects (17-19) and had negligible effects on mouse survival or fertility (20). These inconsistent findings suggest that the functionally relevant substrates of FTO during mammalian development are still unknown.

[0004] Unlike Pcif1 KO, Mettl3 ablation in mice leads to early embryonic lethality (21). Recently, it was found that chromatin-associated regulatory RNAs (carRNAs) can be mRNAs of METTL3. 6 A methylation, METTL3 regulates chromatin state and transcription in mouse embryonic stem cells (mESCs) (22); independent reports have confirmed that carRNA m 6 A's chromatin regulatory role, and also demonstrated that m6 A has a significant impact on endogenous retroviral (ERV) expression and heterochromatin formation (23-25). 6 Whether a subset of A-labeled carRNAs can be physiological substrates of FTO and whether FTO-mediated m 6 Whether A demethylation can regulate chromatin status in mammalian tissues and early in development remains to be seen.

[0005] In 2011, FTO was used as mRNA 6 The discovery of A demethylases has opened a new field in biology to study the regulation of gene expression by dynamic RNA modifications. The present inventors recently made another potentially paradigm-changing discovery using Fto knockout (KO) mESC and mouse models. The present inventors discovered a universal mRNA of non-coding chromatin-associated regulatory RNAs (carRNAs). 6 A methylation. Methylation of carRNA and FTO-mediated demethylation affect transcription by regulating the abundance of carRNA and the chromatin state nearby. On the other hand, the inventors overexpressed FTO in crops such as rice and potatoes and found that FTO overexpression (OE) can increase crop yield by about 50%. Although rice and potatoes are plants of different families, their overgrowth phenotypes are the same, indicating that this process has a certain universality. These findings are of great significance to crop breeding and food safety, and have prompted the inventors to think about whether FTO OE in mice, humans and animals (such as livestock) can promote reproduction and increase yields in animal husbandry.

[0006] It was previously known that transgenic overexpression of FTO in mice causes obesity, but whole-body overexpression mouse models failed to reveal when and where FTO overexpression (OE) leads to the obesity phenotype. Furthermore, transgenic OE, which alters the genomic DNA sequence, raises concerns about the safety of genetic modification. Summary of the Invention

[0007] In general, the present disclosure relates to FTO-mediated m 6 The present disclosure also relates to the discovery that transient FTO overexpression (OE) in early embryos promotes embryonic growth, increases birth weight and adult weight, and improves zygote implantation rate without altering genomic DNA.

[0008] The disclosed methods include, but are not limited to, methods for demethylating LINE1 RNA in cells, methods for altering gene expression levels in cells, methods for increasing chromatin accessibility in cells, methods for optimizing germ cell development, and include 1, 2, 3, 4 or more steps, including any of the following steps: providing an effective amount of fat mass and obesity-related protein (FTO) and / or a nucleic acid molecule encoding FTO to cells, detecting methylation in the cells, and culturing and / or incubating the cells in culture medium and / or buffer.

[0009] The disclosed methods also include, but are not limited to, methods for altering gene expression levels in cells, methods for reducing chromatin accessibility in cells, methods for optimizing germ cell development, and include 1, 2, 3, 4 or more steps, including any of the following steps: providing an effective amount of a FTO inhibitor to the cells, detecting methylation in the cells, and culturing and / or incubating the cells in culture medium and / or buffer.

[0010] The disclosed methods also include methods for increasing zygote implantation in animals, methods for increasing litter size, methods for improving in vitro fertilization processes, methods for increasing litter size and number of litters in animals, methods for increasing zygote implantation rate, methods for increasing birth weight of animals, methods for reducing miscarriage in animals and / or methods for reducing the risk of metabolic diseases, comprising 1, 2, 3, 4, 5, 6, 7 or more steps, including any of the following steps: implanting one or more fertilized cells into the reproductive tract of an animal, monitoring the pregnancy or implantation rate of the animal, detecting hormonal changes in the animal such as HCG levels, detecting demethylation including RNA demethylation in the fertilized cells, and culturing and / or incubating the fertilized cells in culture medium and / or buffer.

[0011] Disclosed herein are methods for demethylating long-interspersed element-1 (LINE1) RNA in cells. The methods may include providing an effective amount of fat mass and obesity-related protein (FTO) and / or a nucleic acid molecule encoding FTO to the cells. Also disclosed are methods for changing gene expression levels in cells, wherein the methods may include providing an effective amount of FTO or a nucleic acid molecule encoding FTO to the cells. Also disclosed are methods for increasing chromatin accessibility in cells, wherein the methods include providing an effective amount of FTO or a nucleic acid molecule encoding FTO to the cells. Also disclosed are methods for optimizing germ cell development, wherein the methods include providing an effective amount of FTO or a nucleic acid molecule encoding FTO to the cells. LINE1 RNA may be a chromatin-associated regulatory RNA. The cells may be any cells, such as germ cells or fertilized cells. The cells may be from any source, such as humans, mice, or livestock animals.

[0012] In some aspects, the method includes providing a nucleic acid encoding FTO to the cell. The nucleic acid provided to the cell may also include a nucleic acid encoding a Cas nuclease and a guide RNA (gRNA). The nucleic acid encoding FTO and the nucleic acid encoding the Cas nuclease and the guide RNA (gRNA) may be located on the same nucleic acid molecule or an independent nucleic acid molecule. In some aspects, the method includes providing FTO protein to the cell. The FTO protein may be provided in the form of a wild-type protein, a mutant protein and / or a fusion protein. The fusion protein may be an FTO protein fused to a Cas nuclease. In the presence of a Cas nuclease fusion, gRNA may also be provided to the cell. The Cas nuclease may be a catalytically inactive Cas13 (dCas13). The gRNA may target any nucleic acid, including LINE1 RNA. The FTO protein or the nucleic acid encoding FTO may be provided in an amount that effectively reduces the amount of histone modification in the cell.

[0013] In certain aspects, the amount of nucleic acid encoding FTO and / or FTO protein provided to the cells is about 1 ng / µL carrier or 1 ng / mL carrier, 2 ng / µL carrier or 2 ng / mL carrier, 3 ng / µL carrier or 3 ng / mL carrier, 4 ng / µL carrier or 4 ng / mL carrier, 5 ng / µL carrier or 5 ng / mL carrier, 6 ng / µL carrier or 6 ng / mL carrier, 7 ng / µL carrier or 7 ng / mL carrier, 8 ng / µL carrier or 8 ng / mL carrier, 9 ng / µL carrier or 9 ng / mL carrier, 10 ng / µL carrier or 10 ng / mL carrier, 11 ng / µL carrier or 11 ng / mL carrier, 12 ng / µL carrier or 12 ng / mL carrier. g / mL carrier, 13ng / µL carrier or 13ng / mL carrier, 14ng / µL carrier or 14ng / mL carrier, 15ng / µL carrier or 15ng / mL carrier, 16ng / µL carrier or 16ng / mL carrier, 17ng / µL carrier or 17ng / mL carrier, 18ng / µL carrier or 18ng / mL carrier, 19ng / µL carrier or 19ng / mL carrier, 20ng / µL carrier or 20ng / mL carrier, 21ng / µL carrier or 21ng / mL carrier, 22ng / µL carrier or 22ng / mL carrier, 23ng / µL carrier or 23ng / mL carrier, 24ng / µL carrier or 24ng / mL carrier, 25 ng / µL Carrier or 25ng / mL Carrier, 26ng / µL Carrier or 26ng / mL Carrier, 27ng / µL Carrier or 27ng / mL Carrier, 28ng / µL Carrier or 28ng / mL Carrier, 29ng / µL Carrier or 29ng / mL Carrier, 30ng / µL Carrier or 30ng / mL Carrier, 31ng / µL Carrier or 31ng / mL Carrier, 32ng / µL Carrier or 32ng / mL Carrier, 33ng / µL Carrier or 33ng / mL Carrier, 34ng / µL Carrier or 34ng / mL Carrier, 35ng / µL Carrier or 35ng / mL Carrier, 36ng / µL Carrier or 36ng / mL Carrier, 37ng / µL Carrier or 3 7ng / mL carrier, 38ng / µL carrier or 38ng / mL carrier, 39ng / µL carrier or 39ng / mL carrier, 40ng / µL carrier or 40ng / mL carrier, 41ng / µL carrier or 41ng / mL carrier, 42ng / µL carrier or 42ng / mL carrier, 43ng / µL carrier or 43ng / mL carrier, 44ng / µL carrier or 44ng / mL carrier, 45ng / µL carrier or 45ng / mL carrier, 46ng / µL carrier or 46ng / mL carrier, 47ng / µL carrier or 47ng / mL carrier, 48ng / µL carrier or 48ng / mL carrier, 49ng / µL carrier or 49ng / mL carrier,50ng / µL Vehicle or 50ng / mL Vehicle, 51ng / µL Vehicle or 51ng / mL Vehicle, 52ng / µL Vehicle or 52ng / mL Vehicle, 53ng / µL Vehicle or 53ng / mL Vehicle, 54ng / µL Vehicle or 54ng / mL Vehicle, 55ng / µL Vehicle or 55ng / mL Vehicle, 56ng / µL Vehicle or 56ng / mL Vehicle, 57ng / µL Vehicle or 57ng / mL Vehicle, 58ng / µL Vehicle or 58ng / mL Vehicle, 59ng / µL Vehicle or 59ng / mL Vehicle, 60ng / µL Vehicle or 60ng / mL Vehicle, 61ng / µL Vehicle or 61ng / mL Vehicle, 62ng / µL Vehicle or 62ng / mL carrier, 63ng / µL carrier or 63ng / mL carrier, 64ng / µL carrier or 64ng / mL carrier, 65ng / µL carrier or 65ng / mL carrier, 66ng / µL carrier or 66ng / mL carrier, 67ng / µL carrier or 67ng / mL carrier, 68ng / µL carrier or 68ng / mL carrier, 69ng / µL carrier or 69ng / mL carrier, 70ng / µL carrier or 70ng / mL carrier, 71ng / µL carrier or 71ng / mL carrier, 72ng / µL carrier or 72ng / mL carrier, 73ng / µL carrier or 73ng / mL carrier, 74ng / µL carrier or 74ng / mL carrier, 75ng / µL Vehicle or 75ng / mL Vehicle, 76ng / µL Vehicle or 76ng / mL Vehicle, 77ng / µL Vehicle or 77ng / mL Vehicle, 78ng / µL Vehicle or 78ng / mL Vehicle, 79ng / µL Vehicle or 79ng / mL Vehicle, 80ng / µL Vehicle or 80ng / mL Vehicle, 81ng / µL Vehicle or 81ng / mL Vehicle, 82ng / µL Vehicle or 82ng / mL Vehicle, 83ng / µL Vehicle or 83ng / mL Vehicle, 84ng / µL Vehicle or 84ng / mL Vehicle, 85ng / µL Vehicle or 85ng / mL Vehicle, 86ng / µL Vehicle or 86ng / mL Vehicle, 87ng / µL Vehicle or 87ng / mL carrier, 88ng / µL carrier or 88ng / mL carrier, 89ng / µL carrier or 89ng / mL carrier, 90ng / µL carrier or 90ng / mL carrier, 91ng / µL carrier or 91ng / mL carrier, 92ng / µL carrier or 92ng / mL carrier, 93ng / µL carrier or 93ng / mL carrier, 94ng / µL carrier or 94ng / mL carrier, 95ng / µL carrier or 95ng / mL carrier, 96ng / µL carrier or 96ng / mL carrier, 97ng / µL 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ng / mL of Vehicle, 120 ng / µL of Vehicle or 120 ng / mL of Vehicle, 121 ng / µL of Vehicle or 121 ng / mL of Vehicle, 122 ng / µL of Vehicle or 122ng / mL carrier, 123ng / µL carrier or 123ng / mL carrier, 124ng / µL carrier or 124ng / mL carrier, 125ng / µL carrier or 125ng / mL carrier, 126ng / µL carrier or 126ng / mL carrier, 127ng / µL carrier or 127ng / mL carrier, 128ng / µL carrier or 128ng / mL carrier, 129ng / µL carrier or 129ng / mL carrier, 130ng / µL carrier or 130ng / mL carrier, 131ng / µL carrier or 131ng / mL carrier, 132ng / µL carrier or 132ng / mL carrier, 133ng / µL carrier or 133ng / mL carrier, 134ng / µL carrier or 134ng / mL carrier, 135ng / µL carrier or 135ng / mL carrier, 136ng / µL carrier or 136ng / mL carrier, 137ng / µL carrier or 137ng / mL carrier, 138ng / µL carrier or 138ng / mL carrier, 139ng / µL carrier or 139ng / mL carrier, 140ng / µL carrier or 140ng / mL carrier, 141ng / µL carrier or 141ng / mL carrier, 142ng / µL carrier or 142ng / mL 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µL Vehicle or 306ng / mL Vehicle, 307ng / µL Vehicle or 307ng / mL Vehicle, 308ng / µL Vehicle or 308ng / mL Vehicle, 309ng / µL Vehicle or 309ng / mL Vehicle, 310ng / µL Vehicle or 310ng / mL Vehicle, 311ng / µL Vehicle or 311ng / mL Vehicle, 312ng / µL Vehicle or 312ng / mL Vehicle, 313ng / µL Vehicle or 313ng / mL carrier, 314ng / µL carrier or 314ng / mL carrier, 315ng / µL carrier or 315ng / mL carrier, 316ng / µL carrier or 316ng / mL carrier, 317ng / µL carrier or 317ng / mL carrier, 318ng / µL carrier or 318ng / mL carrier, 319ng / µL carrier or 319ng / mL carrier, 320ng / µL carrier or 320ng / mL carrier, 321ng / µL carrier or 321ng / mL carrier, 322ng / µL carrier or 322ng / mL carrier, 323ng / µL carrier or 323ng / mL carrier, 324ng / µL carrier or 324ng / mL carrier,325ng / µL carrier or 325ng / mL carrier, 326ng / µL carrier or 326ng / mL carrier, 327ng / µL carrier or 327ng / mL carrier, 328ng / µL carrier or 328ng / mL carrier, 329ng / µL carrier or 329ng / mL carrier, 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mL carrier, 446ng / µL carrier or 446ng / mL carrier, 447ng / µL carrier or 447ng / mL carrier, 448ng / µL carrier or 448ng / mL carrier, 449ng / µL carrier or 449ng / mL carrier, 450ng / µL carrier or 450ng / mL carrier, 451ng / µL carrier or 451ng / mL carrier, 452ng / µL carrier or 452ng / mL carrier, 453ng / µL carrier or 453ng / mL carrier, 454ng / µL carrier or 454ng / mL carrier, 455ng / µL carrier or 455ng / mL carrier, 456ng / µL carrier or 456ng / mL carrier, 457ng / µL carrier or 457ng / mL carrier, 458ng / µL carrier or 458ng / mL carrier, 459ng / µL carrier or 459ng / mL carrier,460ng / µL carrier or 460ng / mL carrier, 461ng / µL carrier or 461ng / mL carrier, 462ng / µL carrier or 462ng / mL carrier, 463ng / µL carrier or 463ng / mL carrier, 464ng / µL carrier or 464ng / mL carrier, 465ng / µL carrier or 465ng / mL carrier, 466ng / µL carrier or 466ng / mL carrier, 467ng / µL carrier or 467ng / mL carrier, 468ng / µL carrier or 468ng / mL carrier, 469ng / µL carrier or 469ng / mL carrier, 470ng / µL carrier or 470ng / mL carrier, 471ng / µL of carrier or 471ng / mL of carrier, 472ng / µL of carrier or 472ng / mL of carrier, 473ng / µL of carrier or 473ng / mL of carrier, 474ng / µL of carrier or 474ng / mL of carrier, 475ng / µL of carrier or 475ng / mL of carrier, 476ng / µL of carrier or 476ng / mL of carrier, 477ng / µL of carrier or 477ng / mL of carrier, 478ng / µL of carrier or 478ng / mL of carrier, 479ng / µL of carrier or 479ng / mL of carrier, 480ng / µL of carrier or 480ng / mL of carrier, 481ng / µL of carrier or 481ng / mL of carrier, 482ng / µL of carrier or 482ng / mL carrier, 483ng / µL carrier or 483ng / mL carrier, 484ng / µL carrier or 484ng / mL carrier, 485ng / µL carrier or 485ng / mL carrier, 486ng / µL carrier or 486ng / mL carrier, 487ng / µL carrier or 487ng / mL carrier, 488ng / µL carrier or 488ng / mL carrier, 489ng / µL carrier or 489ng / mL carrier, 490ng / µL carrier or 490ng / mL carrier, 491ng / µL carrier or 491ng / mL carrier, 492ng / µL carrier or 492ng / mL carrier, 493ng / µL carrier or 493ng / mL carrier, 494ng / µL carrier or 494ng / mL carrier, 495ng / µL carrier or 495ng / mL carrier, 496ng / µL carrier or 496ng / mL carrier, 497ng / µL carrier or 497ng / mL carrier, 498ng / µL carrier or 498ng / mL carrier, 499ng / µL carrier or 499ng / mL carrier, 500ng / µL carrier or 500ng / mL carrier, 501ng / µL carrier or 501ng / mL carrier, 502ng / µL carrier or 502ng / mL carrier, 503ng / µL carrier or 503ng / mL carrier, 504ng / µL carrier or 504ng / mL carrier,505ng / µL carrier or 505ng / mL carrier, 506ng / µL carrier or 506ng / mL carrier, 507ng / µL carrier or 507ng / mL carrier, 508ng / µL carrier or 508ng / mL carrier, 509ng / µL carrier or 509ng / mL carrier, 510ng / µL carrier or 510ng / mL carrier, 511ng / µL carrier or 511ng / mL carrier, 512ng / µL carrier or 512ng / mL carrier, 513ng / µL carrier or 513ng / mL carrier, 514ng / µL carrier or 514ng / mL carrier, 515ng / µL carrier or 515ng / mL carrier, 516ng / µL Vehicle or 516ng / mL Vehicle, 517ng / µL Vehicle or 517ng / mL Vehicle, 518ng / µL Vehicle or 518ng / mL Vehicle, 519ng / µL Vehicle or 519ng / mL Vehicle, 520ng / µL Vehicle or 520ng / mL Vehicle, 521ng / µL Vehicle or 521ng / mL Vehicle, 522ng / µL Vehicle or 522ng / mL Vehicle, 523ng / µL Vehicle or 523ng / mL Vehicle, 524ng / µL Vehicle or 524ng / mL Vehicle, 525ng / µL Vehicle or 525ng / mL Vehicle, 526ng / µL Vehicle or 526ng / mL Vehicle, 527ng / µL Vehicle or 527ng / mL carrier, 528ng / µL carrier or 528ng / mL carrier, 529ng / µL carrier or 529ng / mL carrier, 530ng / µL carrier or 530ng / mL carrier, 531ng / µL carrier or 531ng / mL carrier, 532ng / µL carrier or 532ng / mL carrier, 533ng / µL carrier or 533ng / mL carrier, 534ng / µL carrier or 534ng / mL carrier, 535ng / µL carrier or 535ng / mL carrier, 536ng / µL carrier or 536ng / mL carrier, 537ng / µL carrier or 537ng / mL carrier, 538ng / µL carrier or 538ng / mL carrier, 539ng / µL carrier or 539ng / mL carrier, 540ng / µL carrier or 540ng / mL carrier, 541ng / µL carrier or 541ng / mL carrier, 542ng / µL carrier or 542ng / mL carrier, 543ng / µL carrier or 543ng / mL carrier, 544ng / µL carrier or 544ng / mL carrier, 545ng / µL carrier or 545ng / mL carrier, 546ng / µL carrier or 546ng / mL carrier, 547ng / µL carrier or 547ng / mL carrier, 548ng / µL carrier or 548ng / mL carrier, 549ng / µL carrier or 549ng / mL carrier,550ng / µL carrier or 550ng / mL carrier, 551ng / µL carrier or 551ng / mL carrier, 552ng / µL carrier or 552ng / mL carrier, 553ng / µL carrier or 553ng / mL carrier, 554ng / µL carrier or 554ng / mL carrier, 555ng / µL carrier or 555ng / mL carrier, 556ng / µL carrier or 556ng / mL carrier, 557ng / µL carrier or 557ng / mL carrier, 558ng / µL carrier or 558ng / mL carrier, 559ng / µL carrier or 559ng / mL carrier, 560ng / µL carrier or 560ng / mL carrier, 561ng / µL of carrier or 561ng / mL of carrier, 562ng / µL of carrier or 562ng / mL of carrier, 563ng / µL of carrier or 563ng / mL of carrier, 564ng / µL of carrier or 564ng / mL of carrier, 565ng / µL of carrier or 565ng / mL of carrier, 566ng / µL of carrier or 566ng / mL of carrier, 567ng / µL of carrier or 567ng / mL of carrier, 568ng / µL of carrier or 568ng / mL of carrier, 569ng / µL of carrier or 569ng / mL of carrier, 570ng / µL of carrier or 570ng / mL of carrier, 571ng / µL of carrier or 571ng / mL of carrier, 572ng / µL of carrier or 572ng / mL Vehicle, 573ng / µL Vehicle or 573ng / mL Vehicle, 574ng / µL Vehicle or 574ng / mL Vehicle, 575ng / µL Vehicle or 575ng / mL Vehicle, 576ng / µL Vehicle or 576ng / mL Vehicle, 577ng / µL Vehicle or 577ng / mL Vehicle, 578ng / µL Vehicle or 578ng / mL Vehicle, 579ng / µL Vehicle or 579ng / mL Vehicle, 580ng / µL Vehicle or 580ng / mL Vehicle, 581ng / µL Vehicle or 581ng / mL Vehicle, 582ng / µL Vehicle or 582ng / mL Vehicle, 583ng / µL Vehicle or 583ng / mL carrier, 584ng / µL carrier or 584ng / mL carrier, 585ng / µL carrier or 585ng / mL carrier, 586ng / µL carrier or 586ng / mL carrier, 587ng / µL carrier or 587ng / mL carrier, 588ng / µL carrier or 588ng / mL carrier, 589ng / µL carrier or 589ng / mL carrier, 590ng / µL carrier or 590ng / mL carrier, 591ng / µL carrier or 591ng / mL carrier, 592ng / µL carrier or 592ng / mL carrier, 593ng / µL carrier or 593ng / mL carrier, 594ng / µL carrier or 594ng / mL carrier,595ng / µL carrier or 595ng / mL carrier, 596ng / µL carrier or 596ng / mL carrier, 597ng / µL carrier or 597ng / mL carrier, 598ng / µL carrier or 598ng / mL carrier, 599ng / µL carrier or 599ng / mL carrier, 600ng / µL carrier or 600ng / mL carrier, 601ng / µL carrier or 601ng / mL carrier, 602ng / µL carrier or 602ng / mL carrier, 603ng / µL carrier or 603ng / mL carrier, 604ng / µL carrier or 604ng / mL carrier, 605ng / µL carrier or 605ng / mL carrier, 606ng / µL Vehicle or 606ng / mL Vehicle, 607ng / µL Vehicle or 607ng / mL Vehicle, 608ng / µL Vehicle or 608ng / mL Vehicle, 609ng / µL Vehicle or 609ng / mL Vehicle, 610ng / µL Vehicle or 610ng / mL Vehicle, 611ng / µL Vehicle or 611ng / mL Vehicle, 612ng / µL Vehicle or 612ng / mL Vehicle, 613ng / µL Vehicle or 613ng / mL Vehicle, 614ng / µL Vehicle or 614ng / mL Vehicle, 615ng / µL Vehicle or 615ng / mL Vehicle, 616ng / µL Vehicle or 616ng / mL Vehicle, 617ng / µL Vehicle or 617ng / mL Vehicle 617ng / mL carrier, 618ng / µL carrier or 618ng / mL carrier, 619ng / µL carrier or 619ng / mL carrier, 620ng / µL carrier or 620ng / mL carrier, 621ng / µL carrier or 621ng / mL carrier, 622ng / µL carrier or 622ng / mL carrier, 623ng / µL carrier or 623ng / mL carrier, 624ng / µL carrier or 624ng / mL carrier, 625ng / µL carrier or 625ng / mL carrier, 626ng / µL carrier or 626ng / mL carrier, 627ng / µL carrier or 627ng / mL carrier, 628ng / µL carrier or 628ng / mL carrier, 629ng / µL carrier or 629ng / mL carrier, 630ng / µL carrier or 630ng / mL carrier, 631ng / µL carrier or 631ng / mL carrier, 632ng / µL carrier or 632ng / mL carrier, 633ng / µL carrier or 633ng / mL carrier, 634ng / µL carrier or 634ng / mL carrier, 635ng / µL carrier or 635ng / mL carrier, 636ng / µL carrier or 636ng / mL carrier, 637ng / µL carrier or 637ng / mL carrier, 638ng / µL carrier or 638ng / mL carrier, 639ng / µL carrier or 639ng / mL carrier,640ng / µL carrier or 640ng / mL carrier, 641ng / µL carrier or 641ng / mL carrier, 642ng / µL carrier or 642ng / mL carrier, 643ng / µL carrier or 643ng / mL carrier, 644ng / µL carrier or 644ng / mL carrier, 645ng / µL carrier or 645ng / mL carrier, 646ng / µL carrier or 646ng / mL carrier, 647ng / µL carrier or 647ng / mL carrier, 648ng / µL carrier or 648ng / mL carrier, 649ng / µL carrier or 649ng / mL carrier, 650ng / µL carrier or 650ng / mL carrier, 651ng / µL Vehicle or 651ng / mL Vehicle, 652ng / µL Vehicle or 652ng / mL Vehicle, 653ng / µL Vehicle or 653ng / mL Vehicle, 654ng / µL Vehicle or 654ng / mL Vehicle, 655ng / µL Vehicle or 655ng / mL Vehicle, 656ng / µL Vehicle or 656ng / mL Vehicle, 657ng / µL Vehicle or 657ng / mL Vehicle, 658ng / µL Vehicle or 658ng / mL Vehicle, 659ng / µL Vehicle or 659ng / mL Vehicle, 660ng / µL Vehicle or 660ng / mL Vehicle, 661ng / µL Vehicle or 661ng / mL Vehicle, 662ng / µL Vehicle or 662ng / mL Vehicle, 663ng / µL Vehicle or 663ng / mL Vehicle, 664ng / µL Vehicle or 664ng / mL Vehicle, 665ng / µL Vehicle or 665ng / mL Vehicle, 666ng / µL Vehicle or 666ng / mL Vehicle, 667ng / µL Vehicle or 667ng / mL Vehicle, 668ng / µL Vehicle or 668ng / mL Vehicle, 669ng / µL Vehicle or 669ng / mL Vehicle, 670ng / µL Vehicle or 670ng / mL Vehicle, 671ng / µL Vehicle or 671ng / mL Vehicle, 672ng / µL Vehicle or 672ng / mL Vehicle, 673ng / µL Vehicle or 673ng / mL carrier, 674ng / µL carrier or 674ng / mL carrier, 675ng / µL carrier or 675ng / mL carrier, 676ng / µL carrier or 676ng / mL carrier, 677ng / µL carrier or 677ng / mL carrier, 678ng / µL carrier or 678ng / mL carrier, 679ng / µL carrier or 679ng / mL carrier, 680ng / µL carrier or 680ng / mL carrier, 681ng / µL carrier or 681ng / mL carrier, 682ng / µL carrier or 682ng / mL carrier, 683ng / µL carrier or 683ng / mL carrier, 684ng / µL carrier or 684ng / mL carrier,685ng / µL carrier or 685ng / mL carrier, 686ng / µL carrier or 686ng / mL carrier, 687ng / µL carrier or 687ng / mL carrier, 688ng / µL carrier or 688ng / mL carrier, 689ng / µL carrier or 689ng / mL carrier, 690ng / µL carrier or 690ng / mL carrier, 691ng / µL carrier or 691ng / mL carrier, 692ng / µL carrier or 692ng / mL carrier, 693ng / µL carrier or 693ng / mL carrier, 694ng / µL carrier or 694ng / mL carrier, 695ng / µL carrier or 695ng / mL carrier, 696ng / µL of carrier or 696ng / mL of carrier, 697ng / µL of carrier or 697ng / mL of carrier, 698ng / µL of carrier or 698ng / mL of carrier, 699ng / µL of carrier or 699ng / mL of carrier, 700ng / µL of carrier or 700ng / mL of carrier, 701ng / µL of carrier or 701ng / mL of carrier, 702ng / µL of carrier or 702ng / mL of carrier, 703ng / µL of carrier or 703ng / mL of carrier, 704ng / µL of carrier or 704ng / mL of carrier, 705ng / µL of carrier or 705ng / mL of carrier, 706ng / µL of carrier or 706ng / mL of carrier, 707ng / µL of carrier or 707ng / mL carrier, 708ng / µL carrier or 708ng / mL carrier, 709ng / µL carrier or 709ng / mL carrier, 710ng / µL carrier or 710ng / mL carrier, 711ng / µL carrier or 711ng / mL carrier, 712ng / µL carrier or 712ng / mL carrier, 713ng / µL carrier or 713ng / mL carrier, 714ng / µL carrier or 714ng / mL carrier, 715ng / µL carrier or 715ng / mL carrier, 716ng / µL carrier or 716ng / mL carrier, 717ng / µL carrier or 717ng / mL carrier, 718ng / µL carrier or 718ng / mL carrier, 719ng / µL carrier or 719ng / mL carrier, 720ng / µL carrier or 720ng / mL carrier, 721ng / µL carrier or 721ng / mL carrier, 722ng / µL carrier or 722ng / mL carrier, 723ng / µL carrier or 723ng / mL carrier, 724ng / µL carrier or 724ng / mL carrier, 725ng / µL carrier or 725ng / mL carrier, 726ng / µL carrier or 726ng / mL carrier, 727ng / µL carrier or 727ng / mL carrier, 728ng / µL carrier or 728ng / mL carrier, 729ng / µL carrier or 729ng / mL carrier,730ng / µL carrier or 730ng / mL carrier, 731ng / µL carrier or 731ng / mL carrier, 732ng / µL carrier or 732ng / mL carrier, 733ng / µL carrier or 733ng / mL carrier, 734ng / µL carrier or 734ng / mL carrier, 735ng / µL carrier or 735ng / mL carrier, 736ng / µL carrier or 736ng / mL carrier, 737ng / µL carrier or 737ng / mL carrier, 738ng / µL carrier or 738ng / mL carrier, 739ng / µL carrier or 739ng / mL carrier, 740ng / µL carrier or 740ng / mL carrier, 741ng / µL of carrier or 741ng / mL of carrier, 742ng / µL of carrier or 742ng / mL of carrier, 743ng / µL of carrier or 743ng / mL of carrier, 744ng / µL of carrier or 744ng / mL of carrier, 745ng / µL of carrier or 745ng / mL of carrier, 746ng / µL of carrier or 746ng / mL of carrier, 747ng / µL of carrier or 747ng / mL of carrier, 748ng / µL of carrier or 748ng / mL of carrier, 749ng / µL of carrier or 749ng / mL of carrier, 750ng / µL of carrier or 750ng / mL of carrier, 751ng / µL of carrier or 751ng / mL of carrier, 752ng / µL of carrier or 752ng / mL carrier, 753ng / µL carrier or 753ng / mL carrier, 754ng / µL carrier or 754ng / mL carrier, 755ng / µL carrier or 755ng / mL carrier, 756ng / µL carrier or 756ng / mL carrier, 757ng / µL carrier or 757ng / mL carrier, 758ng / µL carrier or 758ng / mL carrier, 759ng / µL carrier or 759ng / mL carrier, 760ng / µL carrier or 760ng / mL carrier, 761ng / µL carrier or 761ng / mL carrier, 762ng / µL carrier or 762ng / mL carrier, 763ng / µL carrier or 763ng / mL carrier, 764ng / µL carrier or 764ng / mL carrier, 765ng / µL carrier or 765ng / mL carrier, 766ng / µL carrier or 766ng / mL carrier, 767ng / µL carrier or 767ng / mL carrier, 768ng / µL carrier or 768ng / mL carrier, 769ng / µL carrier or 769ng / mL carrier, 770ng / µL carrier or 770ng / mL carrier, 771ng / µL carrier or 771ng / mL carrier, 772ng / µL carrier or 772ng / mL carrier, 773ng / µL carrier or 773ng / mL carrier, 774ng / µL carrier or 774ng / mL carrier,775ng / µL carrier or 775ng / mL carrier, 776ng / µL carrier or 776ng / mL carrier, 777ng / µL carrier or 777ng / mL carrier, 778ng / µL carrier or 778ng / mL carrier, 779ng / µL carrier or 779ng / mL carrier, 780ng / µL carrier or 780ng / mL carrier, 781ng / µL carrier or 781ng / mL carrier, 782ng / µL carrier or 782ng / mL carrier, 783ng / µL carrier or 783ng / mL carrier, 784ng / µL carrier or 784ng / mL carrier, 785ng / µL carrier or 785ng / mL carrier, 786ng / µL carrier or 786ng / mL carrier, 787ng / µL carrier or 787ng / mL carrier, 788ng / µL carrier or 788ng / mL carrier, 789ng / µL carrier or 789ng / mL carrier, 790ng / µL carrier or 790ng / mL carrier, 791ng / µL carrier or 791ng / mL carrier, 792ng / µL carrier or 792ng / mL carrier, 793ng / µL carrier or 793ng / mL carrier, 794ng / µL carrier or 794ng / mL carrier, 795ng / µL carrier or 795ng / mL carrier, 796ng / µL carrier or 796ng / mL carrier, 797ng / µL carrier or 797ng / mL carrier, 798ng / µL carrier or 798ng / mL carrier, 799ng / µL carrier or 799ng / mL carrier, 800ng / µL carrier or 800ng / mL carrier, 801ng / µL carrier or 801ng / mL carrier, 802ng / µL carrier or 802ng / mL carrier, 803ng / µL carrier or 803ng / mL carrier, 804ng / µL carrier or 804ng / mL carrier, 805ng / µL carrier or 805ng / mL carrier, 806ng / µL carrier or 806ng / mL carrier, 807ng / µL carrier or 807ng / mL carrier, 808ng / µL carrier or 808ng / mL carrier, 809ng / µL carrier or 809ng / mL carrier, 810ng / µL carrier or 810ng / mL carrier, 811ng / µL carrier or 811ng / mL carrier, 812ng / µL carrier or 812ng / mL carrier, 813ng / µL carrier or 813ng / mL carrier, 814ng / µL carrier or 814ng / mL carrier, 815ng / µL carrier or 815ng / mL carrier, 816ng / µL carrier or 816ng / mL carrier, 817ng / µL carrier or 817ng / mL carrier, 818ng / µL carrier or 818ng / mL carrier, 819ng / µL carrier or 819ng / mL carrier,820ng / µL carrier or 820ng / mL carrier, 821ng / µL carrier or 821ng / mL carrier, 822ng / µL carrier or 822ng / mL carrier, 823ng / µL carrier or 823ng / mL carrier, 824ng / µL carrier or 824ng / mL carrier, 825ng / µL carrier or 825ng / mL carrier, 826ng / µL carrier or 826ng / mL carrier, 827ng / µL carrier or 827ng / mL carrier, 828ng / µL carrier or 828ng / mL carrier, 829ng / µL carrier or 829ng / mL carrier, 830ng / µL carrier or 830ng / mL carrier, 831ng / µL of carrier or 831ng / mL of carrier, 832ng / µL of carrier or 832ng / mL of carrier, 833ng / µL of carrier or 833ng / mL of carrier, 834ng / µL of carrier or 834ng / mL of carrier, 835ng / µL of carrier or 835ng / mL of carrier, 836ng / µL of carrier or 836ng / mL of carrier, 837ng / µL of carrier or 837ng / mL of carrier, 838ng / µL of carrier or 838ng / mL of carrier, 839ng / µL of carrier or 839ng / mL of carrier, 840ng / µL of carrier or 840ng / mL of carrier, 841ng / µL of carrier or 841ng / mL of carrier, 842ng / µL of carrier or 842ng / mL carrier, 843ng / µL carrier or 843ng / mL carrier, 844ng / µL carrier or 844ng / mL carrier, 845ng / µL carrier or 845ng / mL carrier, 846ng / µL carrier or 846ng / mL carrier, 847ng / µL carrier or 847ng / mL carrier, 848ng / µL carrier or 848ng / mL carrier, 849ng / µL carrier or 849ng / mL carrier, 850ng / µL carrier or 850ng / mL carrier, 851ng / µL carrier or 851ng / mL carrier, 852ng / µL carrier or 852ng / mL carrier, 853ng / µL carrier or 853ng / mL carrier, 854ng / µL carrier or 854ng / mL carrier, 855ng / µL carrier or 855ng / mL carrier, 856ng / µL carrier or 856ng / mL carrier, 857ng / µL carrier or 857ng / mL carrier, 858ng / µL carrier or 858ng / mL carrier, 859ng / µL carrier or 859ng / mL carrier, 860ng / µL carrier or 860ng / mL carrier, 861ng / µL carrier or 861ng / mL carrier, 862ng / µL carrier or 862ng / mL carrier, 863ng / µL carrier or 863ng / mL carrier, 864ng / µL carrier or 864ng / mL carrier,865ng / µL carrier or 865ng / mL carrier, 866ng / µL carrier or 866ng / mL carrier, 867ng / µL carrier or 867ng / mL carrier, 868ng / µL carrier or 868ng / mL carrier, 869ng / µL carrier or 869ng / mL carrier, 870ng / µL carrier or 870ng / mL carrier, 871ng / µL carrier or 871ng / mL carrier, 872ng / µL carrier or 872ng / mL carrier, 873ng / µL carrier or 873ng / mL carrier, 874ng / µL carrier or 874ng / mL carrier, 875ng / µL carrier or 875ng / mL carrier, 876ng / µL of carrier or 876ng / mL of carrier, 877ng / µL of carrier or 877ng / mL of carrier, 878ng / µL of carrier or 878ng / mL of carrier, 879ng / µL of carrier or 879ng / mL of carrier, 880ng / µL of carrier or 880ng / mL of carrier, 881ng / µL of carrier or 881ng / mL of carrier, 882ng / µL of carrier or 882ng / mL of carrier, 883ng / µL of carrier or 883ng / mL of carrier, 884ng / µL of carrier or 884ng / mL of carrier, 885ng / µL of carrier or 885ng / mL of carrier, 886ng / µL of carrier or 886ng / mL of carrier, 887ng / µL of carrier or 887ng / mL of carrier 887ng / mL carrier, 888ng / µL carrier or 888ng / mL carrier, 889ng / µL carrier or 889ng / mL carrier, 890ng / µL carrier or 890ng / mL carrier, 891ng / µL carrier or 891ng / mL carrier, 892ng / µL carrier or 892ng / mL carrier, 893ng / µL carrier or 893ng / mL carrier, 894ng / µL carrier or 894ng / mL carrier, 895ng / µL carrier or 895ng / mL carrier, 896ng / µL carrier or 896ng / mL carrier, 897ng / µL carrier or 897ng / mL carrier, 898ng / µL carrier or 898ng / mL carrier, 899ng / µL carrier or 899ng / mL carrier, 900ng / µL carrier or 900ng / mL carrier, 901ng / µL carrier or 901ng / mL carrier, 902ng / µL carrier or 902ng / mL carrier, 903ng / µL carrier or 903ng / mL carrier, 904ng / µL carrier or 904ng / mL carrier, 905ng / µL carrier or 905ng / mL carrier, 906ng / µL carrier or 906ng / mL carrier, 907ng / µL carrier or 907ng / mL carrier, 908ng / µL carrier or 908ng / mL carrier, 909ng / µL carrier or 909ng / mL carrier,910ng / µL carrier or 910ng / mL carrier, 911ng / µL carrier or 911ng / mL carrier, 912ng / µL carrier or 912ng / mL carrier, 913ng / µL carrier or 913ng / mL carrier, 914ng / µL carrier or 914ng / mL carrier, 915ng / µL carrier or 915ng / mL carrier, 916ng / µL carrier or 916ng / mL carrier, 917ng / µL carrier or 917ng / mL carrier, 918ng / µL carrier or 918ng / mL carrier, 919ng / µL carrier or 919ng / mL carrier, 920ng / µL carrier or 920ng / mL carrier, 921ng / µL carrier or 921ng / mL carrier, 922ng / µL carrier or 922ng / mL carrier, 923ng / µL carrier or 923ng / mL carrier, 924ng / µL carrier or 924ng / mL carrier, 925ng / µL carrier or 925ng / mL carrier, 926ng / µL carrier or 926ng / mL carrier, 927ng / µL carrier or 927ng / mL carrier, 928ng / µL carrier or 928ng / mL carrier, 929ng / µL carrier or 929ng / mL carrier, 930ng / µL carrier or 930ng / mL carrier, 931ng / µL carrier or 931ng / mL carrier, 932ng / µL carrier or 932ng / mL carrier, 933ng / µL carrier or 933ng / mL carrier, 934ng / µL carrier or 934ng / mL carrier, 935ng / µL carrier or 935ng / mL carrier, 936ng / µL carrier or 936ng / mL carrier, 937ng / µL carrier or 937ng / mL carrier, 938ng / µL carrier or 938ng / mL carrier, 939ng / µL carrier or 939ng / mL carrier, 940ng / µL carrier or 940ng / mL carrier, 941ng / µL carrier or 941ng / mL carrier, 942ng / µL carrier or 942ng / mL carrier, 943ng / µL carrier or 943ng / mL carrier, 944ng / µL carrier or 944ng / mL carrier, 945ng / µL carrier or 945ng / mL carrier, 946ng / µL carrier or 946ng / mL carrier, 947ng / µL carrier or 947ng / mL carrier, 948ng / µL carrier or 948ng / mL carrier, 949ng / µL carrier or 949ng / mL carrier, 950ng / µL carrier or 950ng / mL carrier, 951ng / µL carrier or 951ng / mL carrier, 952ng / µL carrier or 952ng / mL carrier, 953ng / µL carrier or 953ng / mL carrier, 954ng / µL carrier or 954ng / mL carrier,955ng / µL carrier or 955ng / mL carrier, 956ng / µL carrier or 956ng / mL carrier, 957ng / µL carrier or 957ng / mL carrier, 958ng / µL carrier or 958ng / mL carrier, 959ng / µL carrier or 959ng / mL carrier, 960ng / µL carrier or 960ng / mL carrier, 961ng / µL carrier or 961ng / mL carrier, 962ng / µL carrier or 962ng / mL carrier, 963ng / µL carrier or 963ng / mL carrier, 964ng / µL carrier or 964ng / mL carrier, 965ng / µL carrier or 965ng / mL carrier, 966ng / µL carrier or 966ng / mL carrier, 967ng / µL carrier or 967ng / mL carrier, 968ng / µL carrier or 968ng / mL carrier, 969ng / µL carrier or 969ng / mL carrier, 970ng / µL carrier or 970ng / mL carrier, 971ng / µL carrier or 971ng / mL carrier, 972ng / µL carrier or 972ng / mL carrier, 973ng / µL carrier or 973ng / mL carrier, 974ng / µL carrier or 974ng / mL carrier, 975ng / µL carrier or 975ng / mL carrier, 976ng / µL carrier or 976ng / mL carrier, 977ng / µL carrier or 977ng / mL carrier, 978ng / µL carrier or 978ng / mL carrier, 979ng / µL carrier or 979ng / mL carrier, 980ng / µL carrier or 980ng / mL carrier, 981ng / µL carrier or 981ng / mL carrier, 982ng / µL carrier or 982ng / mL carrier, 983ng / µL carrier or 983ng / mL carrier, 984ng / µL carrier or 984ng / mL carrier, 985ng / µL carrier or 985ng / mL carrier, 986ng / µL carrier or 986ng / mL carrier, 987ng / µL carrier or 987ng / mL carrier, 988ng / µL carrier or 988ng / mL carrier, 989ng / µL carrier or 989ng / mL carrier, 990ng / µL carrier or 990ng / mL carrier, 991ng / µL carrier or 991ng / mL carrier, 992ng / µL carrier or 992ng / mL carrier, 993ng / µL carrier or 993ng / mL carrier, 994ng / µL carrier or 994ng / mL carrier, 995ng / µL carrier or 995ng / mL carrier, 996ng / µL carrier or 996ng / mL carrier, 997ng / µL carrier or 997ng / mL carrier, 998ng / µL carrier or 998ng / mL carrier, 999ng / µL carrier or 999ng / mL carrier,1000 ng / µL carrier or 1000 ng / mL carrier (or higher than 1000 ng / µL carrier or 1000 ng / mL carrier or any range derived therefrom). The carrier can be any carrier suitable for contacting the protein and / or nucleic acid, such as water (including nuclease-free water), buffer solution, salt solution, DMSO or other suitable liquid. The carrier can be a cell culture medium. The nucleic acid and / or protein can be provided in a carrier such as a liposome or virus that can introduce the nucleic acid and / or protein into the cell.

[0014] Also disclosed are methods for increasing LINE1 RNA methylation in cells. The methods may include providing an effective amount of an FTO inhibitor to the cells. Also disclosed are methods for changing gene expression levels in cells, wherein the methods include providing an effective amount of an FTO inhibitor to the cells. Also disclosed are methods for reducing chromatin accessibility in cells, wherein the methods include providing an effective amount of an FTO inhibitor to the cells. Also disclosed are methods for optimizing germ cell development, wherein the methods include providing an effective amount of an FTO inhibitor to the cells. The FTO inhibitor may be any one of FB23, FB23-2, CS1, CS2, and / or Dac51, or a combination thereof. The inhibitor may be provided in an amount effective to reduce chromatin accessibility in the cells. It is particularly contemplated that any of these inhibitors may be excluded in the embodiments. In certain aspects, the FTO inhibitor is administered at 1 ng / μL carrier or 1 ng / mL carrier or 1 mg / mL carrier, 2 ng / μL carrier or 2 ng / mL carrier or 2 mg / mL carrier, 3 ng / μL carrier or 3 ng / mL carrier or 3 mg / mL carrier, 4 ng / μL carrier or 4 ng / mL carrier or 4 mg / mL carrier, 5 ng / μL carrier or 5 ng / mL carrier or 5 mg / mL carrier, 6 ng / μL carrier or 6 ng / mL carrier or 6 mg / mL carrier, 7 ng / μL carrier or 7 ng / mL carrier or 7 mg / mL carrier, 8 ng / μL carrier or 8 ng / mL carrier or 8 mg / mL carrier, 9 ng / μL carrier or 9 ng / mL carrier or 9 mg / mL carrier, 10 ng / μL carrier or 10 ng / mL carrier or 10 mg / mL carrier, 11 ng / μL carrier or 11 ng / mL carrier or 11 mg / mL carrier, 12 ng / μL carrier or 12 ng / mL carrier or 12 mg / mL carrier, 13 ng / μL carrier or 14 ng / mL carrier or 15 mg / mL carrier, 14 ng / μL carrier or 15 ng / mL carrier or 16 mg / mL carrier, 15 ng / μL carrier or 16 ng / mL carrier or 17 mg / mL carrier, 16 ng / μL carrier or 17 ng / mL carrier or 18 mg / mL carrier, 17 ng / μL carrier or 18 ng / mL carrier or 19 mg / mL carrier, 18 ng / μL carrier or 19 ng / mL carrier or 20 mg / mL carrier, 19 ng / μL carrier or 20 ng / mL carrier or 21 mg / mL carrier, L carrier or 13ng / mL carrier or 13mg / mL carrier, 14ng / μL carrier or 14ng / mL carrier or 14mg / mL carrier, 15ng / μL carrier or 15ng / mL carrier or 15mg / mL carrier, 16ng / μL carrier or 16ng / mL carrier or 16mg / mL carrier, 17ng / μL carrier or 17ng / mL carrier or 17mg / mL carrier, 18ng / μL carrier or 18ng / mL carrier or 18mg / mL carrier, 19ng / μL carrier or 19ng / mL carrier or 19ng / mL carrier, 10ng / μL carrier or 10ng / mL carrier or 10mg / mL carrier, 11ng / μL carrier or 11ng / mL carrier or 11mg / mL carrier, 12ng / μL carrier or 12ng / mL carrier or 12mg / mL carrier, 13ng / μL carrier or 13ng / mL carrier or 13mg / mL carrier, 14ng / μL carrier or 14ng / mL carrier or 14mg / mL carrier, 15ng / μL carrier or 15ng / mL carrier or 15mg / mL carrier, 16ng / μL carrier or 16ng / mL carrier or 16mg / mL carrier, g / μL carrier or 19 ng / mL carrier or 19 mg / mL carrier, 20 ng / μL carrier or 20 ng / mL carrier or 20 mg / mL carrier, 21 ng / μL carrier or 21 ng / mL carrier or 21 mg / mL carrier, 22 ng / μL carrier or 22 ng / mL carrier or 22 mg / mL carrier, 23 ng / μL carrier or 23 ng / mL carrier or 23 mg / mL carrier, 24 ng / μL carrier or 24 ng / mL carrier or 24 mg / mL carrier,25 ng / μL carrier or 25 ng / mL carrier or 25 mg / mL carrier, 26 ng / μL carrier or 26 ng / mL carrier or 26 mg / mL carrier, 27 ng / μL carrier or 27 ng / mL carrier or 27 mg / mL carrier, 28 ng / μL carrier or 28 ng / mL carrier or 28 mg / mL carrier, 29 ng / μL carrier or 29 ng / mL carrier or 29 mg / mL carrier, 30 ng / μL carrier or 30 ng / mL carrier or 30 mg / mL carrier, 31 ng / μL carrier or 31 ng / mL carrier or 31 mg / mL carrier, 32 ng / μL carrier or 32 ng / mL carrier or 32 mg / mL carrier, 33 ng / μL carrier or 34 ng / mL carrier or 35 mg / mL carrier, carrier or 33 ng / mL carrier or 33 mg / mL carrier, 34 ng / μL carrier or 34 ng / mL carrier or 34 mg / mL carrier, 35 ng / μL carrier or 35 ng / mL carrier or 35 mg / mL carrier, 36 ng / μL carrier or 36 ng / mL carrier or 36 mg / mL carrier, 37 ng / μL carrier or 37 ng / mL carrier or 37 mg / mL carrier, 38 ng / μL carrier or 38 ng / mL carrier or 38 mg / mL carrier, 39 ng / μL carrier or 39 ng / mL carrier or 39 mg / mL carrier, 40 ng / μL carrier or 40 ng / mL carrier or 40 mg / mL carrier, 41 ng / μL carrier or 41 ng / mL carrier or 41 mg / mL carrier, 42 ng / μL carrier or 42 ng / mL carrier or 42 mg / mL carrier, 43 ng / μL carrier or 43 ng / mL carrier or 43 mg / mL carrier, 44 ng / μL carrier or 44 ng / mL carrier or 44 mg / mL carrier, 45 ng / μL carrier or 45 ng / mL carrier or 45 mg / mL carrier, 46 ng / μL carrier or 46 ng / mL carrier or 46 mg / mL carrier, 47 ng / μL carrier or 47 ng / mL carrier or 47 mg / mL carrier, 48 ng / μL carrier or 48 ng / mL carrier or 48 mg / mL carrier, 49 ng / μL carrier or 49 ng / mL carrier or 49 mg / mL carrier, 50 ng / μL carrier or 50 ng / mL carrier or 50 mg / mL carrier, 51 ng / μL carrier or 51 ng / mL carrier or 51 mg / mL carrier, 52 ng / μL carrier or 52 ng / mL carrier or 52 mg / mL carrier, 53 ng / μL carrier or 53 ng / mL carrier or 53 mg / mL carrier, 54 ng / μL carrier or 54 ng / mL carrier or 54 mg / mL carrier, 55 ng / μL carrier or 55 ng / mL carrier or 55 mg / mL carrier, 56 ng / μL carrier or 56 ng / mL carrier or 56 mg / mL carrier, 57 ng / μL carrier or 57 ng / mL carrier or 57 mg / mL carrier,58 ng / μL carrier or 58 ng / mL carrier or 58 mg / mL carrier, 59 ng / μL carrier or 59 ng / mL carrier or 59 mg / mL carrier, 60 ng / μL carrier or 60 ng / mL carrier or 60 mg / mL carrier, 61 ng / μL carrier or 61 ng / mL carrier or 61 mg / mL carrier, 62 ng / μL carrier or 62 ng / mL carrier or 62 mg / mL carrier, 63 ng / μL carrier or 63 ng / mL carrier or 63 mg / mL carrier, 64 ng / μL carrier or 64 ng / mL carrier or 64 mg / mL carrier, 65 ng / μL carrier or 65 ng / mL carrier or 65 mg / mL carrier, 66 ng / μL carrier or 67 ng / mL carrier or 68 mg / mL carrier, Carrier or 66ng / mL carrier or 66mg / mL carrier, 67ng / μL carrier or 67ng / mL carrier or 67mg / mL carrier, 68ng / μL carrier or 68ng / mL carrier or 68mg / mL carrier, 69ng / μL carrier or 69ng / mL carrier or 69mg / mL carrier, 70ng / μL carrier or 70ng / mL carrier or 70mg / mL carrier, 71ng / μL carrier or 71ng / mL carrier or 71mg / mL carrier, 72ng / μL carrier or 72ng / mL carrier or 72mg / mL carrier, 73ng / μL carrier or 73ng / mL carrier or 73mg / mL carrier, 74ng / μL carrier or 74ng / mL carrier or 74ng / mL carrier. mL carrier or 74 mg / mL carrier, 75 ng / μL carrier or 75 ng / mL carrier or 75 mg / mL carrier, 76 ng / μL carrier or 76 ng / mL carrier or 76 mg / mL carrier, 77 ng / μL carrier or 77 ng / mL carrier or 77 mg / mL carrier, 78 ng / μL carrier or 78 ng / mL carrier or 78 mg / mL carrier, 79 ng / μL carrier or 79 ng / mL carrier or 79 mg / mL carrier, 80 ng / μL carrier or 80 ng / mL carrier or 80 mg / mL carrier, 81 ng / μL carrier or 81 ng / mL carrier or 81 mg / mL carrier, 82 ng / μL carrier or 82 ng / mL carrier or 82 mg / mL carrier, 83 ng / μL carrier or 83 ng / mL carrier or 83 mg / mL carrier, 84 ng / μL carrier or 84 ng / mL carrier or 84 mg / mL carrier, 85 ng / μL carrier or 85 ng / mL carrier or 85 mg / mL carrier, 86 ng / μL carrier or 86 ng / mL carrier or 86 mg / mL carrier, 87 ng / μL carrier or 87 ng / mL carrier or 87 mg / mL carrier, 88 ng / μL carrier or 88 ng / mL carrier or 88 mg / mL carrier, 89 ng / μL carrier or 89 ng / mL carrier or 89 mg / mL carrier, 90 ng / μL carrier or 90 ng / mL carrier or 90 mg / mL carrier,91 ng / μL carrier or 91 ng / mL carrier or 91 mg / mL carrier, 92 ng / μL carrier or 92 ng / mL carrier or 92 mg / mL carrier, 93 ng / μL carrier or 93 ng / mL carrier or 93 mg / mL carrier, 94 ng / μL carrier or 94 ng / mL carrier or 94 mg / mL carrier, 95 ng / μL carrier or 95 ng / mL carrier or 95 mg / mL carrier, 96 ng / μL carrier or 96 ng / mL carrier or 96 mg / mL carrier, 97 ng / μL carrier or 97 ng / mL carrier or 97 mg / mL carrier, 98 ng / μL carrier or 98 ng / mL carrier or 98 mg / mL carrier, 99 ng / μL carrier or 99 ng / mL carrier or 99 mg / mL carrier, 100 ng / μL carrier or 100 ng / mL carrier or 100 mg / mL carrier, 101 ng / μL carrier or 101 ng / mL carrier or 101 mg / mL carrier, 102 ng / μL carrier or 102 ng / mL carrier or 102 mg / mL carrier, 103 ng / μL carrier or 103 ng / mL carrier or 103 mg / mL carrier, 104 ng / μL carrier or 104 ng / mL carrier or 104 mg / mL carrier, 105 ng / μL carrier or 105 ng / mL carrier or 105 mg / mL carrier, 106 ng / μL carrier or 106 ng / mL carrier or 106 mg / mL carrier, 107 ng / μL carrier or 107 ng / mL carrier or 107 mg / mL carrier, 108 ng / μL carrier or 108 ng / mL carrier or 108 mg / mL carrier, 109 ng / μL carrier or 109 ng / mL carrier or 109 mg / mL carrier, 110 ng / μL carrier or 110 ng / mL carrier or 110 mg / mL carrier, 111 ng / μL carrier or 111 ng / mL carrier or 111 mg / mL carrier, 112 ng / μL carrier or 112 ng / mL carrier or 112 mg / mL carrier, 113 ng / μL carrier or 113 ng / mL carrier or 113 mg / mL carrier, 114 ng / μL carrier or 114 n g / mL carrier or 114 mg / mL carrier, 115 ng / μL carrier or 115 ng / mL carrier or 115 mg / mL carrier, 116 ng / μL carrier or 116 ng / mL carrier or 116 mg / mL carrier, 117 ng / μL carrier or 117 ng / mL carrier or 117 mg / mL carrier, 118 ng / μL carrier or 118 ng / mL carrier or 118 mg / mL carrier, 119 ng / μL carrier or 119 ng / mL carrier or 119 mg / mL carrier, 120 ng / μL carrier or 120 ng / mL carrier or 120 mg / mL carrier, 121 ng / μL carrier or 121 ng / mL carrier or 121 mg / mL carrier,122 ng / μL carrier or 122 ng / mL carrier or 122 mg / mL carrier, 123 ng / μL carrier or 123 ng / mL carrier or 123 mg / mL carrier, 124 ng / μL carrier or 124 ng / mL carrier or 124 mg / mL carrier, 125 ng / μL carrier or 125 ng / mL carrier or 125 mg / mL carrier, 126 ng / μL carrier or 126 ng / mL carrier or 126 mg / mL carrier, 127 ng / μL carrier or 127 ng / mL carrier or 127 mg / mL carrier, 128 ng / μL carrier or 128 ng / mL carrier or 128 mg / mL carrier, 129 ng / μL carrier or 129 ng / mL carrier or 129 mg / mL carrier, 130 ng / μL carrier or 130 ng / mL carrier or 130 mg / mL carrier, 131 ng / μL carrier or 131 ng / mL carrier or 131 mg / mL carrier, 132 ng / μL carrier or 132 ng / mL carrier or 132 mg / mL carrier, 133 ng / μL carrier or 133 ng / mL carrier or 133 mg / mL carrier, 134 ng / μL carrier or 134 ng / mL carrier or 134 mg / mL carrier, 135 ng / μL carrier or 135 ng / mL carrier or 135 mg / mL carrier, 136 ng / μL carrier or 136 ng / mL carrier or 136 mg / mL carrier, 137 ng / μL carrier or 137 ng / mL carrier or 137 mg / mL carrier, 138 ng / μL carrier or 138 ng / mL carrier or 138 mg / mL carrier, 139 ng / μL carrier or 139 ng / mL carrier or 139 mg / mL carrier, 140 ng / μL carrier or 140 ng / mL carrier or 140 mg / mL carrier, 141 ng / μL carrier or 141 ng / mL carrier or 141 mg / mL carrier, 142 ng / μL carrier or 142 ng / mL carrier or 142 mg / mL carrier, 143 ng / μL carrier or 143 ng / mL carrier or 143 mg / mL carrier, 144 ng / μL carrier or 144 ng / mL carrier or 144mg / mL carrier, 145ng / μL carrier or 145ng / mL carrier or 145mg / mL carrier, 146ng / μL carrier or 146ng / mL carrier or 146mg / mL carrier, 147ng / μL carrier or 147ng / mL carrier or 147mg / mL carrier, 148ng / μL carrier or 148ng / mL carrier or 148mg / mL carrier, 149ng / μL carrier or 149ng / mL carrier or 149mg / mL carrier, 150ng / μL carrier or 150ng / mL carrier or 150mg / mL carrier, 151ng / μL carrier or 151ng / mL carrier or 151mg / mL carrier,152 ng / μL carrier or 152 ng / mL carrier or 152 mg / mL carrier, 153 ng / μL carrier or 153 ng / mL carrier or 153 mg / mL carrier, 154 ng / μL carrier or 154 ng / mL carrier or 154 mg / mL carrier, 155 ng / μL carrier or 155 ng / mL carrier or 155 mg / mL carrier, 156 ng / μL carrier or 156 ng / mL carrier or 156 mg / mL carrier, 157 ng / μL carrier or 157 ng / mL carrier or 157 mg / mL carrier, 158 ng / μL carrier or 158 ng / mL carrier or 158 mg / mL carrier, 159 ng / μL carrier or 159 ng / mL carrier Carrier or 159mg / mL carrier, 160ng / μL carrier or 160ng / mL carrier or 160mg / mL carrier, 161ng / μL carrier or 161ng / mL carrier or 161mg / mL carrier, 162ng / μL carrier or 162ng / mL carrier or 162mg / mL carrier, 163ng / μL carrier or 163ng / mL carrier or 163mg / mL carrier, 164ng / μL carrier or 164ng / mL carrier or 164mg / mL carrier, 165ng / μL carrier or 165ng / mL carrier or 165mg / mL carrier, 166ng / μL carrier or 166ng / mL carrier or 166mg / mL carrier, 167ng / μL carrier or 167ng / mL carrier or 167ng / mL carrier, 168ng / μL carrier or 168ng / mL carrier or 168mg / mL carrier, 169ng / μL carrier or 169ng / mL carrier or 169mg / mL carrier, 170ng / μL carrier or 170ng / mL carrier or 170mg / mL carrier, 171ng / μL carrier or 171ng / mL carrier or 171mg / mL carrier, 172ng / μL carrier or 172ng / mL carrier or 172mg / mL carrier, / μL carrier or 167ng / mL carrier or 167mg / mL carrier, 168ng / μL carrier or 168ng / mL carrier or 168mg / mL carrier, 169ng / μL carrier or 169ng / mL carrier or 169mg / mL carrier, 170ng / μL carrier or 170ng / mL carrier or 170mg / mL carrier, 171ng / μL carrier or 171ng / mL carrier or 171mg / mL carrier, 172ng / μL carrier or 172ng / mL carrier or 172mg / mL carrier, 173ng / μL carrier or 173ng / mL carrier or 173mg / mL carrier, 174ng / μL carrier or 174ng / mL carrier or 174mg / mL carrier, 175ng / μL carrier or 175ng / mL carrier or 175mg / mL carrier, 4 mg / mL carrier, 175 ng / μL carrier or 175 ng / mL carrier or 175 mg / mL carrier, 176 ng / μL carrier or 176 ng / mL carrier or 176 mg / mL carrier, 177 ng / μL carrier or 177 ng / mL carrier or 177 mg / mL carrier, 178 ng / μL carrier or 178 ng / mL carrier or 178 mg / mL carrier, 179 ng / μL carrier or 179 ng / mL carrier or 179 mg / mL carrier, 180 ng / μL carrier or 180 ng / mL carrier or 180 mg / mL carrier, 181 ng / μL carrier or 181 ng / mL carrier or 181 mg / mL carrier, 182 ng / μL carrier,

[0015] or 182 ng / mL vehicle or 182 mg / mL vehicle, 183 ng / μL vehicle or 183 ng / mL vehicle or 183 mg / mL vehicle, 184 ng / μL vehicle or 184 ng / mL vehicle or 184 mg / mL vehicle, 185 ng / μL vehicle or 185 ng / mL vehicle or 185 mg / mL vehicle, 186 ng / μL vehicle or 186 ng / mL vehicle or 186 mg / mL vehicle, 187 ng / μL vehicle or 187 ng / mL vehicle or 187 mg / mL vehicle, 188 ng / μL vehicle or 188 ng / mL vehicle or 188 mg / mL vehicle, 189 ng / μL vehicle or 189 ng / mL vehicle or 180 mg / mL vehicle, 9 mg / mL carrier, 190 ng / μL carrier or 190 ng / mL carrier or 190 mg / mL carrier, 191 ng / μL carrier or 191 ng / mL carrier or 191 mg / mL carrier, 192 ng / μL carrier or 192 ng / mL carrier or 192 mg / mL carrier, 193 ng / μL carrier or 193 ng / mL carrier or 193 mg / mL carrier, 194 ng / μL carrier or 194 ng / mL carrier or 194 mg / mL carrier, 195 ng / μL carrier or 195 ng / mL carrier or 195 mg / mL carrier, 196 ng / μL carrier or 196 ng / mL carrier or 196 mg / mL carrier, 197 ng / μL carrier or 197 ng / mL carrier or 197 mg / mL carrier, 198 ng / μL carrier or 199 ng / mL carrier or 199 mg / mL carrier, 199 ng / μL carrier or 199 ng / mL carrier or 199 mg / mL carrier, / μL carrier or 197 ng / mL carrier or 197 mg / mL carrier, 198 ng / μL carrier or 198 ng / mL carrier or 198 mg / mL carrier, 199 ng / μL carrier or 199 ng / mL carrier or 199 mg / mL carrier, 200 ng / μL carrier or 200 ng / mL carrier or 200 mg / mL carrier, 201 ng / μL carrier or 201 ng / mL carrier or 201 mg / mL carrier, 202 ng / μL carrier or 202 ng / mL carrier or 202 mg / mL carrier, 203 ng / μL carrier or 203 ng / mL carrier or 203 mg / mL carrier, 204 ng / μL carrier or 204 ng / mL carrier carrier or 204 mg / mL carrier, 205 ng / μL carrier or 205 ng / mL carrier or 205 mg / mL carrier, 206 ng / μL carrier or 206 ng / mL carrier or 206 mg / mL carrier, 207 ng / μL carrier or 207 ng / mL carrier or 207 mg / mL carrier, 208 ng / μL carrier or 208 ng / mL carrier or 208 mg / mL carrier, 209 ng / μL carrier or 209 ng / mL carrier or 209 mg / mL carrier, 210 ng / μL carrier or 210 ng / mL carrier or 210 mg / mL carrier, 211 ng / μL carrier or 211 ng / mL carrier or 211 mg / mL carrier,212 ng / μL carrier or 212 ng / mL carrier or 212 mg / mL carrier, 213 ng / μL carrier or 213 ng / mL carrier or 213 mg / mL carrier, 214 ng / μL carrier or 214 ng / mL carrier or 214 mg / mL carrier, 215 ng / μL carrier or 215 ng / mL carrier or 215 mg / mL carrier, 216 ng / μL carrier or 216 ng / mL carrier or 216 mg / mL carrier, 217 ng / μL carrier or 217 ng / mL carrier or 217 mg / mL carrier, 218 ng / μL carrier or 218 ng / mL carrier or 218 mg / mL carrier, 219 ng / μL carrier or 219 ng / mL carrier or 219 mg / mL carrier, 220 ng / μL carrier or 220 ng / mL carrier or 220 mg / mL carrier, 221 ng / μL carrier or 221 ng / mL carrier or 221 mg / mL carrier, 222 ng / μL carrier or 222 ng / mL carrier or 222 mg / mL carrier, 223 ng / μL carrier or 223 ng / mL carrier or 223 mg / mL carrier, 224 ng / μL carrier or 224 ng / mL carrier or 224 mg / mL carrier, 225 ng / μL carrier or 225 ng / mL carrier or 225 mg / mL carrier, 226 ng / μL carrier or 226 ng / mL carrier or 226 mg / mL carrier, 227 ng / μL carrier or 227 ng / mL carrier or 227 mg / mL carrier, 228 ng / μL carrier or 228 ng / mL carrier or 228 mg / mL carrier, 229 ng / μL carrier or 229 ng / mL carrier or 229 mg / mL carrier, 230 ng / μL carrier or 230 ng / mL carrier or 230 mg / mL carrier, 231 ng / μL carrier or 231 ng / mL carrier or 231 mg / mL carrier, 232 ng / μL carrier or 232 ng / mL carrier or 232 mg / mL carrier, 233 ng / μL carrier or 233 ng / mL carrier or 233 mg / mL carrier, 234 ng / μL carrier or 234 ng / mL carrier or 234 mg / mL carrier, 235 ng / μL carrier or 235 ng / mL carrier or 235 mg / mL carrier, 236 ng / μL carrier or 236 ng / mL carrier or 236 mg / mL carrier, 237 ng / μL carrier or 237 ng / mL carrier or 237 mg / mL carrier, 238 ng / μL carrier or 238 ng / mL carrier or 238 mg / mL carrier, 239 ng / μL carrier or 239 ng / mL carrier or 239 mg / mL carrier, 240 ng / μL carrier or 240 ng / mL carrier or 240 mg / mL carrier, 241 ng / μL carrier or 241 ng / mL carrier or 241 mg / mL carrier,242 ng / μL carrier or 242 ng / mL carrier or 242 mg / mL carrier, 243 ng / μL carrier or 243 ng / mL carrier or 243 mg / mL carrier, 244 ng / μL carrier or 244 ng / mL carrier or 244 mg / mL carrier, 245 ng / μL carrier or 245 ng / mL carrier or 245 mg / mL carrier, 246 ng / μL carrier or 246 ng / mL carrier or 246 mg / mL carrier, 247 ng / μL carrier or 247 ng / mL carrier or 247 mg / mL carrier, 248 ng / μL carrier or 248 ng / mL carrier or 248 mg / mL carrier, 249 ng / μL carrier or 249 ng / mL carrier or 249 mg / mL carrier, 250 ng / μL carrier or 250 ng / mL carrier or 250 mg / mL carrier, 251 ng / μL carrier or 251 ng / mL carrier or 251 mg / mL carrier, 252 ng / μL carrier or 252 ng / mL carrier or 252 mg / mL carrier, 253 ng / μL carrier or 253 ng / mL carrier or 253 mg / mL carrier, 254 ng / μL carrier or 254 ng / mL carrier or 254 mg / mL carrier, 255 ng / μL carrier or 255 ng / mL carrier or 255 mg / mL carrier, 256 ng / μL carrier or 256 ng / mL carrier or 256 mg / mL carrier, 257 ng / μL carrier or 257 ng / mL carrier or 257 mg / mL carrier, 258 ng / μL carrier or 258 ng / mL carrier or 258 mg / mL carrier, 259 ng / μL carrier or 259 ng / mL carrier or 259 mg / mL carrier, 260 ng / μL carrier or 260 ng / mL carrier or 260 mg / mL carrier, 261 ng / μL carrier or 261 ng / mL carrier or 261 mg / mL carrier, 262 ng / μL carrier or 262 ng / mL carrier or 262 mg / mL carrier, 263 ng / μL carrier or 263 ng / mL carrier or 263 mg / mL carrier, 264 ng / μL carrier or 264 ng / mL carrier or 264mg / mL carrier, 265ng / μL carrier or 265ng / mL carrier or 265mg / mL carrier, 266ng / μL carrier or 266ng / mL carrier or 266mg / mL carrier, 267ng / μL carrier or 267ng / mL carrier or 267mg / mL carrier, 268ng / μL carrier or 268ng / mL carrier or 268mg / mL carrier, 269ng / μL carrier or 269ng / mL carrier or 269mg / mL carrier, 270ng / μL carrier or 270ng / mL carrier or 270mg / mL carrier, 271ng / μL carrier or 271ng / mL carrier or 271mg / mL carrier,272 ng / μL carrier or 272 ng / mL carrier or 272 mg / mL carrier, 273 ng / μL carrier or 273 ng / mL carrier or 273 mg / mL carrier, 274 ng / μL carrier or 274 ng / mL carrier or 274 mg / mL carrier, 275 ng / μL carrier or 275 ng / mL carrier or 275 mg / mL carrier, 276 ng / μL carrier or 276 ng / mL carrier or 276 mg / mL carrier, 277 ng / μL carrier or 277 ng / mL carrier or 277 mg / mL carrier, 278 ng / μL carrier or 278 ng / mL carrier or 278 mg / mL carrier, 279 ng / μL carrier or 279 ng / mL carrier or 279mg / mL carrier, 280ng / μL carrier or 280ng / mL carrier or 280mg / mL carrier, 281ng / μL carrier or 281ng / mL carrier or 281mg / mL carrier, 282ng / μL carrier or 282ng / mL carrier or 282mg / mL carrier, 283ng / μL carrier or 283ng / mL carrier or 283mg / mL carrier, 284ng / μL carrier or 284ng / mL carrier or 284mg / mL carrier, 285ng / μL carrier or 285ng / mL carrier or 285mg / mL carrier, 286ng / μL carrier or 286ng / mL carrier or 286mg / mL carrier, 287 ng / μL carrier or 287 ng / mL carrier or 287 mg / mL carrier, 288 ng / μL carrier or 288 ng / mL carrier or 288 mg / mL carrier, 289 ng / μL carrier or 289 ng / mL carrier or 289 mg / mL carrier, 290 ng / μL carrier or 290 ng / mL carrier or 290 mg / mL carrier, 291 ng / μL carrier or 291 ng / mL carrier or 291 mg / mL carrier, 292 ng / μL carrier or 292 ng / mL carrier or 292 mg / mL carrier, 293 ng / μL carrier or 293 ng / mL carrier or 293 mg / mL carrier, 294 ng / μL carrier or 294 ng / mL carrier or 294mg / mL carrier, 295ng / μL carrier or 295ng / mL carrier or 295mg / mL carrier, 296ng / μL carrier or 296ng / mL carrier or 296mg / mL carrier, 297ng / μL carrier or 297ng / mL carrier or 297mg / mL carrier, 298ng / μL carrier or 298ng / mL carrier or 298mg / mL carrier, 299ng / μL carrier or 299ng / mL carrier or 299mg / mL carrier, 300ng / μL carrier or 300ng / mL carrier or 300mg / mL carrier, 301ng / μL carrier or 301ng / mL carrier or 301mg / mL carrier,302 ng / μL carrier or 302 ng / mL carrier or 302 mg / mL carrier, 303 ng / μL carrier or 303 ng / mL carrier or 303 mg / mL carrier, 304 ng / μL carrier or 304 ng / mL carrier or 304 mg / mL carrier, 305 ng / μL carrier or 305 ng / mL carrier or 305 mg / mL carrier, 306 ng / μL carrier or 306 ng / mL carrier or 306 mg / mL carrier, 307 ng / μL carrier or 307 ng / mL carrier or 307 mg / mL carrier, 308 ng / μL carrier or 308 ng / mL carrier or 308 mg / mL carrier, 309 ng / μL carrier or 309 ng / mL carrier or 309mg / mL carrier, 310ng / μL carrier or 310ng / mL carrier or 310mg / mL carrier, 311ng / μL carrier or 311ng / mL carrier or 311mg / mL carrier, 312ng / μL carrier or 312ng / mL carrier or 312mg / mL carrier, 313ng / μL carrier or 313ng / mL carrier or 313mg / mL carrier, 314ng / μL carrier or 314ng / mL carrier or 314mg / mL carrier, 315ng / μL carrier or 315ng / mL carrier or 315mg / mL carrier, 316ng / μL carrier or 316ng / mL carrier or 316mg / mL carrier, 317 ng / μL carrier or 317 ng / mL carrier or 317 mg / mL carrier, 318 ng / μL carrier or 318 ng / mL carrier or 318 mg / mL carrier, 319 ng / μL carrier or 319 ng / mL carrier or 319 mg / mL carrier, 320 ng / μL carrier or 320 ng / mL carrier or 320 mg / mL carrier, 321 ng / μL carrier or 321 ng / mL carrier or 321 mg / mL carrier, 322 ng / μL carrier or 322 ng / mL carrier or 322 mg / mL carrier, 323 ng / μL carrier or 323 ng / mL carrier or 323 mg / mL carrier, 324 ng / μL carrier or 324 ng / mL carrier or 324 mg / mL carrier, 325 ng / μL carrier or 325 ng / mL carrier or 325 mg / mL carrier, 326 ng / μL carrier or 326 ng / mL carrier or 326 mg / mL carrier, 327 ng / μL carrier or 327 ng / mL carrier or 327 mg / mL carrier, 328 ng / μL carrier or 328 ng / mL carrier or 328 mg / mL carrier, 329 ng / μL carrier or 329 ng / mL carrier or 329 mg / mL carrier, 330 ng / μL carrier or 330 ng / mL carrier or 330 mg / mL carrier, 331 ng / μL carrier or 331 ng / mL carrier or 331 mg / mL carrier,332 ng / μL carrier or 332 ng / mL carrier or 332 mg / mL carrier, 333 ng / μL carrier or 333 ng / mL carrier or 333 mg / mL carrier, 334 ng / μL carrier or 334 ng / mL carrier or 334 mg / mL carrier, 335 ng / μL carrier or 335 ng / mL carrier or 335 mg / mL carrier, 336 ng / μL carrier or 336 ng / mL carrier or 336 mg / mL carrier, 337 ng / μL carrier or 337 ng / mL carrier or 337 mg / mL carrier, 338 ng / μL carrier or 338 ng / mL carrier or 338 mg / mL carrier, 339 ng / μL carrier or 339 ng / mL carrier or 339mg / mL carrier, 340ng / μL carrier or 340ng / mL carrier or 340mg / mL carrier, 341ng / μL carrier or 341ng / mL carrier or 341mg / mL carrier, 342ng / μL carrier or 342ng / mL carrier or 342mg / mL carrier, 343ng / μL carrier or 343ng / mL carrier or 343mg / mL carrier, 344ng / μL carrier or 344ng / mL carrier or 344mg / mL carrier, 345ng / μL carrier or 345ng / mL carrier or 345mg / mL carrier, 346ng / μL carrier or 346ng / mL carrier or 346mg / mL carrier, 347 ng / μL carrier or 347 ng / mL carrier or 347 mg / mL carrier, 348 ng / μL carrier or 348 ng / mL carrier or 348 mg / mL carrier, 349 ng / μL carrier or 349 ng / mL carrier or 349 mg / mL carrier, 350 ng / μL carrier or 350 ng / mL carrier or 350 mg / mL carrier, 351 ng / μL carrier or 351 ng / mL carrier or 351 mg / mL carrier, 352 ng / μL carrier or 352 ng / mL carrier or 352 mg / mL carrier, 353 ng / μL carrier or 353 ng / mL carrier or 353 mg / mL carrier, 354 ng / μL carrier or 354 ng / mL carrier or 354mg / mL carrier, 355ng / μL carrier or 355ng / mL carrier or 355mg / mL carrier, 356ng / μL carrier or 356ng / mL carrier or 356mg / mL carrier, 357ng / μL carrier or 357ng / mL carrier or 357mg / mL carrier, 358ng / μL carrier or 358ng / mL carrier or 358mg / mL carrier, 359ng / μL carrier or 359ng / mL carrier or 359mg / mL carrier, 360ng / μL carrier or 360ng / mL carrier or 360mg / mL carrier, 361ng / μL carrier or 361ng / mL carrier or 361mg / mL carrier,362 ng / μL carrier or 362 ng / mL carrier or 362 mg / mL carrier, 363 ng / μL carrier or 363 ng / mL carrier or 363 mg / mL carrier, 364 ng / μL carrier or 364 ng / mL carrier or 364 mg / mL carrier, 365 ng / μL carrier or 365 ng / mL carrier or 365 mg / mL carrier, 366 ng / μL carrier or 366 ng / mL carrier or 366 mg / mL carrier, 367 ng / μL carrier or 367 ng / mL carrier or 367 mg / mL carrier, 368 ng / μL carrier or 368 ng / mL carrier or 368 mg / mL carrier, 369 ng / μL carrier or 369 ng / mL carrier or 369mg / mL carrier, 370ng / μL carrier or 370ng / mL carrier or 370mg / mL carrier, 371ng / μL carrier or 371ng / mL carrier or 371mg / mL carrier, 372ng / μL carrier or 372ng / mL carrier or 372mg / mL carrier, 373ng / μL carrier or 373ng / mL carrier or 373mg / mL carrier, 374ng / μL carrier or 374ng / mL carrier or 374mg / mL carrier, 375ng / μL carrier or 375ng / mL carrier or 375mg / mL carrier, 376ng / μL carrier or 376ng / mL carrier or 376mg / mL carrier, 377 ng / μL carrier or 377 ng / mL carrier or 377 mg / mL carrier, 378 ng / μL carrier or 378 ng / mL carrier or 378 mg / mL carrier, 379 ng / μL carrier or 379 ng / mL carrier or 379 mg / mL carrier, 380 ng / μL carrier or 380 ng / mL carrier or 380 mg / mL carrier, 381 ng / μL carrier or 381 ng / mL carrier or 381 mg / mL carrier, 382 ng / μL carrier or 382 ng / mL carrier or 382 mg / mL carrier, 383 ng / μL carrier or 383 ng / mL carrier or 383 mg / mL carrier, 384 ng / μL carrier or 384 ng / mL carrier or 384mg / mL carrier, 385ng / μL carrier or 385ng / mL carrier or 385mg / mL carrier, 386ng / μL carrier or 386ng / mL carrier or 386mg / mL carrier, 387ng / μL carrier or 387ng / mL carrier or 387mg / mL carrier, 388ng / μL carrier or 388ng / mL carrier or 388mg / mL carrier, 389ng / μL carrier or 389ng / mL carrier or 389mg / mL carrier, 390ng / μL carrier or 390ng / mL carrier or 390mg / mL carrier, 391ng / μL carrier or 391ng / mL carrier or 391mg / mL carrier,392 ng / μL carrier or 392 ng / mL carrier or 392 mg / mL carrier, 393 ng / μL carrier or 393 ng / mL carrier or 393 mg / mL carrier, 394 ng / μL carrier or 394 ng / mL carrier or 394 mg / mL carrier, 395 ng / μL carrier or 395 ng / mL carrier or 395 mg / mL carrier, 396 ng / μL carrier or 396 ng / mL carrier or 396 mg / mL carrier, 397 ng / μL carrier or 397 ng / mL carrier or 397 mg / mL carrier, 398 ng / μL carrier or 398 ng / mL carrier or 398 mg / mL carrier, 399 ng / μL carrier or 399 ng / mL carrier or 399mg / mL carrier, 400ng / μL carrier or 400ng / mL carrier or 400mg / mL carrier, 401ng / μL carrier or 401ng / mL carrier or 401mg / mL carrier, 402ng / μL carrier or 402ng / mL carrier or 402mg / mL carrier, 403ng / μL carrier or 403ng / mL carrier or 403mg / mL carrier, 404ng / μL carrier or 404ng / mL carrier or 404mg / mL carrier, 405ng / μL carrier or 405ng / 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753 ng / mL carrier or 753 mg / mL carrier, 754 ng / μL carrier or 754 ng / mL carrier or 754 mg / mL carrier, 755 ng / μL carrier or 755 ng / mL carrier or 755 mg / mL carrier, 756 ng / μL carrier or 756 ng / mL carrier or 756 mg / mL carrier, 757 ng / μL carrier or 757 ng / mL carrier or 757 mg / mL carrier, 758 ng / μL carrier or 758 ng / mL carrier or 758 mg / mL carrier, 759 ng / μL carrier or 759 ng / mL carrier or 759mg / mL carrier, 760ng / μL carrier or 760ng / mL carrier or 760mg / mL carrier, 761ng / μL carrier or 761ng / mL carrier or 761mg / mL carrier, 762ng / μL carrier or 762ng / mL carrier or 762mg / mL carrier, 763ng / μL carrier or 763ng / mL carrier or 763mg / mL carrier, 764ng / μL carrier or 764ng / mL carrier or 764mg / mL carrier, 765ng / μL carrier or 765ng / mL carrier or 765mg / mL carrier, 766ng / μL carrier or 766ng / mL carrier or 766mg / mL carrier, 767 ng / μL carrier or 767 ng / mL carrier or 767 mg / mL carrier, 768 ng / μL carrier or 768 ng / mL carrier or 768 mg / mL carrier, 769 ng / μL carrier or 769 ng / mL carrier or 769 mg / mL carrier, 770 ng / μL carrier or 770 ng / mL carrier or 770 mg / mL carrier, 771 ng / μL carrier or 771 ng / mL carrier or 771 mg / mL carrier, 772 ng / μL carrier or 772 ng / mL carrier or 772 mg / mL carrier, 773 ng / μL carrier or 773 ng / mL carrier or 773 mg / mL carrier, 774 ng / μL carrier or 774 ng / mL carrier or 774mg / mL carrier, 775ng / μL carrier or 775ng / mL carrier or 775mg / mL carrier, 776ng / μL carrier or 776ng / mL carrier or 776mg / mL carrier, 777ng / μL carrier or 777ng / mL carrier or 777mg / mL carrier, 778ng / μL carrier or 778ng / mL carrier or 778mg / mL carrier, 779ng / μL carrier or 779ng / mL carrier or 779mg / mL carrier, 780ng / μL carrier or 780ng / mL carrier or 780mg / mL carrier, 781ng / μL carrier or 781ng / mL carrier or 781mg / mL carrier,782 ng / μL carrier or 782 ng / mL carrier or 782 mg / mL carrier, 783 ng / μL carrier or 783 ng / mL carrier or 783 mg / mL carrier, 784 ng / μL carrier or 784 ng / mL carrier or 784 mg / mL carrier, 785 ng / μL carrier or 785 ng / mL carrier or 785 mg / mL carrier, 786 ng / μL carrier or 786 ng / mL carrier or 786 mg / mL carrier, 787 ng / μL carrier or 787 ng / mL carrier or 787 mg / mL carrier, 788 ng / μL carrier or 788 ng / mL carrier or 788 mg / mL carrier, 789 ng / μL carrier or 789 ng / mL carrier or 789mg / mL carrier, 790ng / μL carrier or 790ng / mL carrier or 790mg / mL carrier, 791ng / μL carrier or 791ng / mL carrier or 791mg / mL carrier, 792ng / μL carrier or 792ng / mL carrier or 792mg / mL carrier, 793ng / μL carrier or 793ng / mL carrier or 793mg / mL carrier, 794ng / μL carrier or 794ng / mL carrier or 794mg / mL carrier, 795ng / μL carrier or 795ng / mL carrier or 795mg / mL carrier, 796ng / μL carrier or 796ng / mL carrier or 796mg / mL carrier, 797 ng / μL carrier or 797 ng / mL carrier or 797 mg / mL carrier, 798 ng / μL carrier or 798 ng / mL carrier or 798 mg / mL carrier, 799 ng / μL carrier or 799 ng / mL carrier or 799 mg / mL carrier, 800 ng / μL carrier or 800 ng / mL carrier or 800 mg / mL carrier, 801 ng / μL carrier or 801 ng / mL carrier or 801 mg / mL carrier, 802 ng / μL carrier or 802 ng / mL carrier or 802 mg / mL carrier, 803 ng / μL carrier or 803 ng / mL carrier or 803 mg / mL carrier, 804 ng / μL carrier or 804 ng / mL carrier or 804mg / mL carrier, 805ng / μL carrier or 805ng / mL carrier or 805mg / mL carrier, 806ng / μL carrier or 806ng / mL carrier or 806mg / mL carrier, 807ng / μL carrier or 807ng / mL carrier or 807mg / mL carrier, 808ng / μL carrier or 808ng / mL carrier or 808mg / mL carrier, 809ng / μL carrier or 809ng / mL carrier or 809mg / mL carrier, 810ng / μL carrier or 810ng / mL carrier or 810mg / mL carrier, 811ng / μL carrier or 811ng / mL carrier or 811mg / mL carrier,812 ng / μL carrier or 812 ng / mL carrier or 812 mg / mL carrier, 813 ng / μL carrier or 813 ng / mL carrier or 813 mg / mL carrier, 814 ng / μL carrier or 814 ng / mL carrier or 814 mg / mL carrier, 815 ng / μL carrier or 815 ng / mL carrier or 815 mg / mL carrier, 816 ng / μL carrier or 816 ng / mL carrier or 816 mg / mL carrier, 817 ng / μL carrier or 817 ng / mL carrier or 817 mg / mL carrier, 818 ng / μL carrier or 818 ng / mL carrier or 818 mg / mL carrier, 819 ng / μL carrier or 819 ng / mL carrier or 819 mg / mL carrier, 820 ng / μL carrier or 820 ng / mL carrier or 820 mg / mL carrier, 821 ng / μL carrier or 821 ng / mL carrier or 821 mg / mL carrier, 822 ng / μL carrier or 822 ng / mL carrier or 822 mg / mL carrier, 823 ng / μL carrier or 823 ng / mL carrier or 823 mg / mL carrier, 824 ng / μL carrier or 824 ng / mL carrier or 824 mg / mL carrier, 825 ng / μL carrier or 825 ng / mL carrier or 825 mg / mL carrier, 826 ng / μL carrier or 826 ng / mL carrier or 826 mg / mL carrier, 827 ng / μL carrier or 827 ng / mL carrier or 827 mg / mL carrier, 828 ng / μL carrier or 828 ng / mL carrier or 828 mg / mL carrier, 829 ng / μL carrier or 829 ng / mL carrier or 829 mg / mL carrier, 830 ng / μL carrier or 830 ng / mL carrier or 830 mg / mL carrier, 831 ng / μL carrier or 831 ng / mL carrier or 831 mg / mL carrier, 832 ng / μL carrier or 832 ng / mL carrier or 832 mg / mL carrier, 833 ng / μL carrier or 833 ng / mL carrier or 833 mg / mL carrier, 834 ng / μL carrier or 834 ng / mL carrier or 834 mg / mL carrier, 835 ng / μL carrier or 835 ng / mL carrier or 835 mg / mL carrier, 836 ng / μL carrier or 836 ng / mL carrier or 836 mg / mL carrier, 837 ng / μL carrier or 837 ng / mL carrier or 837 mg / mL carrier, 838 ng / μL carrier or 838 ng / mL carrier or 838 mg / mL carrier, 839 ng / μL carrier or 839 ng / mL carrier or 839 mg / mL carrier, 840 ng / μL carrier or 840 ng / mL carrier or 840 mg / mL carrier, 841 ng / μL carrier or 841 ng / mL carrier or 841 mg / mL carrier,842 ng / μL carrier or 842 ng / mL carrier or 842 mg / mL carrier, 843 ng / μL carrier or 843 ng / mL carrier or 843 mg / mL carrier, 844 ng / μL carrier or 844 ng / mL carrier or 844 mg / mL carrier, 845 ng / μL carrier or 845 ng / mL carrier or 845 mg / mL carrier, 846 ng / μL carrier or 846 ng / mL carrier or 846 mg / mL carrier, 847 ng / μL carrier or 847 ng / mL carrier or 847 mg / mL carrier, 848 ng / μL carrier or 848 ng / mL carrier or 848 mg / mL carrier, 849 ng / μL carrier or 849 ng / mL carrier or 849mg / mL carrier, 850ng / μL carrier or 850ng / mL carrier or 850mg / mL carrier, 851ng / μL carrier or 851ng / mL carrier or 851mg / mL carrier, 852ng / μL carrier or 852ng / mL carrier or 852mg / mL carrier, 853ng / μL carrier or 853ng / mL carrier or 853mg / mL carrier, 854ng / μL carrier or 854ng / mL carrier or 854mg / mL carrier, 855ng / μL carrier or 855ng / mL carrier or 855mg / mL carrier, 856ng / μL carrier or 856ng / mL carrier or 856mg / mL carrier, 857 ng / μL carrier or 857 ng / mL carrier or 857 mg / mL carrier, 858 ng / μL carrier or 858 ng / mL carrier or 858 mg / mL carrier, 859 ng / μL carrier or 859 ng / mL carrier or 859 mg / mL carrier, 860 ng / μL carrier or 860 ng / mL carrier or 860 mg / mL carrier, 861 ng / μL carrier or 861 ng / mL carrier or 861 mg / mL carrier, 862 ng / μL carrier or 862 ng / mL carrier or 862 mg / mL carrier, 863 ng / μL carrier or 863 ng / mL carrier or 863 mg / mL carrier, 864 ng / μL carrier or 864 ng / mL carrier or 864mg / mL carrier, 865ng / μL carrier or 865ng / mL carrier or 865mg / mL carrier, 866ng / μL carrier or 866ng / mL carrier or 866mg / mL carrier, 867ng / μL carrier or 867ng / mL carrier or 867mg / mL carrier, 868ng / μL carrier or 868ng / mL carrier or 868mg / mL carrier, 869ng / μL carrier or 869ng / mL carrier or 869mg / mL carrier, 870ng / μL carrier or 870ng / mL carrier or 870mg / mL carrier, 871ng / μL carrier or 871ng / mL carrier or 871mg / mL carrier,872 ng / μL carrier or 872 ng / mL carrier or 872 mg / mL carrier, 873 ng / μL carrier or 873 ng / mL carrier or 873 mg / mL carrier, 874 ng / μL carrier or 874 ng / mL carrier or 874 mg / mL carrier, 875 ng / μL carrier or 875 ng / mL carrier or 875 mg / mL carrier, 876 ng / μL carrier or 876 ng / mL carrier or 876 mg / mL carrier, 877 ng / μL carrier or 877 ng / mL carrier or 877 mg / mL carrier, 878 ng / μL carrier or 878 ng / mL carrier or 878 mg / mL carrier, 879 ng / μL carrier or 879 ng / mL carrier or 879mg / mL carrier, 880ng / μL carrier or 880ng / mL carrier or 880mg / mL carrier, 881ng / μL carrier or 881ng / mL carrier or 881mg / mL carrier, 882ng / μL carrier or 882ng / mL carrier or 882mg / mL carrier, 883ng / μL carrier or 883ng / mL carrier or 883mg / mL carrier, 884ng / μL carrier or 884ng / mL carrier or 884mg / mL carrier, 885ng / μL carrier or 885ng / mL carrier or 885mg / mL carrier, 886ng / μL carrier or 886ng / mL carrier or 886mg / mL carrier, 887 ng / μL carrier or 887 ng / mL carrier or 887 mg / mL carrier, 888 ng / μL carrier or 888 ng / mL carrier or 888 mg / mL carrier, 889 ng / μL carrier or 889 ng / mL carrier or 889 mg / mL carrier, 890 ng / μL carrier or 890 ng / mL carrier or 890 mg / mL carrier, 891 ng / μL carrier or 891 ng / mL carrier or 891 mg / mL carrier, 892 ng / μL carrier or 892 ng / mL carrier or 892 mg / mL carrier, 893 ng / μL carrier or 893 ng / mL carrier or 893 mg / mL carrier, 894 ng / μL carrier or 894 ng / mL carrier or 894mg / mL carrier, 895ng / μL carrier or 895ng / mL carrier or 895mg / mL carrier, 896ng / μL carrier or 896ng / mL carrier or 896mg / mL carrier, 897ng / μL carrier or 897ng / mL carrier or 897mg / mL carrier, 898ng / μL carrier or 898ng / mL carrier or 898mg / mL carrier, 899ng / μL carrier or 899ng / mL carrier or 899mg / mL carrier, 900ng / μL carrier or 900ng / mL carrier or 900mg / mL carrier, 901ng / μL carrier or 901ng / mL carrier or 901mg / mL carrier,902 ng / μL carrier or 902 ng / mL carrier or 902 mg / mL carrier, 903 ng / μL carrier or 903 ng / mL carrier or 903 mg / mL carrier, 904 ng / μL carrier or 904 ng / mL carrier or 904 mg / mL carrier, 905 ng / μL carrier or 905 ng / mL carrier or 905 mg / mL carrier, 906 ng / μL carrier or 906 ng / mL carrier or 906 mg / mL carrier, 907 ng / μL carrier or 907 ng / mL carrier or 907 mg / mL carrier, 908 ng / μL carrier or 908 ng / mL carrier or 908 mg / mL carrier, 909 ng / μL carrier or 909 ng / mL carrier or 909mg / mL carrier, 910ng / μL carrier or 910ng / mL carrier or 910mg / mL carrier, 911ng / μL carrier or 911ng / mL carrier or 911mg / mL carrier, 912ng / μL carrier or 912ng / mL carrier or 912mg / mL carrier, 913ng / μL carrier or 913ng / mL carrier or 913mg / mL carrier, 914ng / μL carrier or 914ng / mL carrier or 914mg / mL carrier, 915ng / μL carrier or 915ng / mL carrier or 915mg / mL carrier, 916ng / μL carrier or 916ng / mL carrier or 916mg / mL carrier, 917 ng / μL carrier or 917 ng / mL carrier or 917 mg / mL carrier, 918 ng / μL carrier or 918 ng / mL carrier or 918 mg / mL carrier, 919 ng / μL carrier or 919 ng / mL carrier or 919 mg / mL carrier, 920 ng / μL carrier or 920 ng / mL carrier or 920 mg / mL carrier, 921 ng / μL carrier or 921 ng / mL carrier or 921 mg / mL carrier, 922 ng / μL carrier or 922 ng / mL carrier or 922 mg / mL carrier, 923 ng / μL carrier or 923 ng / mL carrier or 923 mg / mL carrier, 924 ng / μL carrier or 924 ng / mL carrier or 924 mg / mL carrier, 925 ng / μL carrier or 925 ng / mL carrier or 925 mg / mL carrier, 926 ng / μL carrier or 926 ng / mL carrier or 926 mg / mL carrier, 927 ng / μL carrier or 927 ng / mL carrier or 927 mg / mL carrier, 928 ng / μL carrier or 928 ng / mL carrier or 928 mg / mL carrier, 929 ng / μL carrier or 929 ng / mL carrier or 929 mg / mL carrier, 930 ng / μL carrier or 930 ng / mL carrier or 930 mg / mL carrier, 931 ng / μL carrier or 931 ng / mL carrier or 931 mg / mL carrier,932 ng / μL carrier or 932 ng / mL carrier or 932 mg / mL carrier, 933 ng / μL carrier or 933 ng / mL carrier or 933 mg / mL carrier, 934 ng / μL carrier or 934 ng / mL carrier or 934 mg / mL carrier, 935 ng / μL carrier or 935 ng / mL carrier or 935 mg / mL carrier, 936 ng / μL carrier or 936 ng / mL carrier or 936 mg / mL carrier, 937 ng / μL carrier or 937 ng / mL carrier or 937 mg / mL carrier, 938 ng / μL carrier or 938 ng / mL carrier or 938 mg / mL carrier, 939 ng / μL carrier or 939 ng / mL carrier or 939mg / mL carrier, 940ng / μL carrier or 940ng / mL carrier or 940mg / mL carrier, 941ng / μL carrier or 941ng / mL carrier or 941mg / mL carrier, 942ng / μL carrier or 942ng / mL carrier or 942mg / mL carrier, 943ng / μL carrier or 943ng / mL carrier or 943mg / mL carrier, 944ng / μL carrier or 944ng / mL carrier or 944mg / mL carrier, 945ng / μL carrier or 945ng / mL carrier or 945mg / mL carrier, 946ng / μL carrier or 946ng / mL carrier or 946mg / mL carrier, 947 ng / μL carrier or 947 ng / mL carrier or 947 mg / mL carrier, 948 ng / μL carrier or 948 ng / mL carrier or 948 mg / mL carrier, 949 ng / μL carrier or 949 ng / mL carrier or 949 mg / mL carrier, 950 ng / μL carrier or 950 ng / mL carrier or 950 mg / mL carrier, 951 ng / μL carrier or 951 ng / mL carrier or 951 mg / mL carrier, 952 ng / μL carrier or 952 ng / mL carrier or 952 mg / mL carrier, 953 ng / μL carrier or 953 ng / mL carrier or 953 mg / mL carrier, 954 ng / μL carrier or 954 ng / mL carrier or 954mg / mL carrier, 955ng / μL carrier or 955ng / mL carrier or 955mg / mL carrier, 956ng / μL carrier or 956ng / mL carrier or 956mg / mL carrier, 957ng / μL carrier or 957ng / mL carrier or 957mg / mL carrier, 958ng / μL carrier or 958ng / mL carrier or 958mg / mL carrier, 959ng / μL carrier or 959ng / mL carrier or 959mg / mL carrier, 960ng / μL carrier or 960ng / mL carrier or 960mg / mL carrier, 961ng / μL carrier or 961ng / mL carrier or 961mg / mL carrier,962 ng / μL carrier or 962 ng / mL carrier or 962 mg / mL carrier, 963 ng / μL carrier or 963 ng / mL carrier or 963 mg / mL carrier, 964 ng / μL carrier or 964 ng / mL carrier or 964 mg / mL carrier, 965 ng / μL carrier or 965 ng / mL carrier or 965 mg / mL carrier, 966 ng / μL carrier or 966 ng / mL carrier or 966 mg / mL carrier, 967 ng / μL carrier or 967 ng / mL carrier or 967 mg / mL carrier, 968 ng / μL carrier or 968 ng / mL carrier or 968 mg / mL carrier, 969 ng / μL carrier or 969 ng / mL carrier or 969mg / mL carrier, 970ng / μL carrier or 970ng / mL carrier or 970mg / mL carrier, 971ng / μL carrier or 971ng / mL carrier or 971mg / mL carrier, 972ng / μL carrier or 972ng / mL carrier or 972mg / mL carrier, 973ng / μL carrier or 973ng / mL carrier or 973mg / mL carrier, 974ng / μL carrier or 974ng / mL carrier or 974mg / mL carrier, 975ng / μL carrier or 975ng / mL carrier or 975mg / mL carrier, 976ng / μL carrier or 976ng / mL carrier or 976mg / mL carrier, 977 ng / μL carrier or 977 ng / mL carrier or 977 mg / mL carrier, 978 ng / μL carrier or 978 ng / mL carrier or 978 mg / mL carrier, 979 ng / μL carrier or 979 ng / mL carrier or 979 mg / mL carrier, 980 ng / μL carrier or 980 ng / mL carrier or 980 mg / mL carrier, 981 ng / μL carrier or 981 ng / mL carrier or 981 mg / mL carrier, 982 ng / μL carrier or 982 ng / mL carrier or 982 mg / mL carrier, 983 ng / μL carrier or 983 ng / mL carrier or 983 mg / mL carrier, 984 ng / μL carrier or 984 ng / mL carrier or 984mg / mL carrier, 985ng / μL carrier or 985ng / mL carrier or 985mg / mL carrier, 986ng / μL carrier or 986ng / mL carrier or 986mg / mL carrier, 987ng / μL carrier or 987ng / mL carrier or 987mg / mL carrier, 988ng / μL carrier or 988ng / mL carrier or 988mg / mL carrier, 989ng / μL carrier or 989ng / mL carrier or 989mg / mL carrier, 990ng / μL carrier or 990ng / mL carrier or 990mg / mL carrier, 991ng / μL carrier or 991ng / mL carrier or 991mg / mL carrier,992 ng / μL carrier or 992 ng / mL carrier or 992 mg / mL carrier, 993 ng / μL carrier or 993 ng / mL carrier or 993 mg / mL carrier, 994 ng / μL carrier or 994 ng / mL carrier or 994 mg / mL carrier, 995 ng / μL carrier or 995 ng / mL carrier or 995 mg / mL carrier, 996 ng / μL carrier or 996 ng / mL carrier or 996 mg / mL carrier, 997 ng / μL carrier or 998 ng / mL carrier or 999 mg / mL carrier, 998 ng / μL carrier or 999 ng / mL carrier or 999 mg / mL carrier, 999 ng / μL carrier or 999 ng / mL carrier or 999 mg / mL carrier, 991 ng / μL carrier or 991 ng / mL carrier or 999 mg / mL carrier, 991 ng / μL carrier or 999 ng / mL carrier or 999 mg / mL carrier, 992 ng / μL carrier or 992 ng / mL carrier or 992 mg / mL carrier, 993 ng / μL carrier or 993 ng / mL carrier or 993 mg / mL carrier, 994 ng / μL carrier or 994 ng / mL carrier or 994 mg / mL carrier, The carrier may be provided at 97 ng / mL carrier or 997 mg / mL carrier, 998 ng / μL carrier or 998 ng / mL carrier or 998 mg / mL carrier, 999 ng / μL carrier or 999 ng / mL carrier or 999 mg / mL carrier, 1000 ng / μL carrier or 1000 ng / mL carrier or 1000 mg / mL carrier (or above 1000 ng / μL carrier or 1000 ng / mL carrier or 1000 mg / mL carrier or any range derivable therefrom). The carrier may be any carrier suitable for contacting proteins and / or nucleic acids, such as water (including nuclease-free water), a buffered solution, a saline solution, DMSO, or other suitable liquid. The carrier may be a cell culture medium. The inhibitor may be provided in an amount effective to increase the amount of histone modification in the cell. The inhibitor may be provided in an amount effective to alter the expression of a LINE1 element and / or a 2C gene. The cell may be any cell, such as an oocyte or a fertilized cell. The cells can be from any source, such as human, mouse, or livestock animals.

[0016] Disclosed herein is a method for increasing zygote implantation in animals. Also disclosed are methods for increasing litter size, methods for improving in vitro fertilization processes, methods for increasing litter size and number of litters in animals, and / or methods for increasing zygote implantation rates. The method may include implanting one or more fertilized cells that have been contacted with exogenous fat mass and obesity-related protein (FTO) and / or nucleic acid molecules encoding FTO that can transiently overexpress exogenous FTO before implantation into the fertilized cells into the animal body. When the FTO protein and / or nucleic acid encoding FTO is introduced into the cell, the fertilized cell can be at any developmental stage. When the FTO protein and / or nucleic acid encoding FTO is introduced, the fertilized cell can be in the zygote development stage, blastocyst stage, morula stage, or embryonic stage. Also disclosed are methods for introducing the FTO protein and / or FTO nucleic acid into the cell before cell fertilization, including introducing into germ cells or gametes, such as eggs or sperm.

[0017] Fertilized cells can be implanted when they are at a stage suitable for implantation, including at the blastocyst stage of development.

[0018] The amount of FTO protein and / or nucleic acid encoding FTO used to contact the cells can be an amount determined by one skilled in the art to be sufficient to affect the disclosed results. The amount of FTO protein and / or nucleic acid encoding FTO used to contact the cells can be 1 ng / µL carrier or 1 ng / mL carrier, 2 ng / µL carrier or 2 ng / mL carrier, 3 ng / µL carrier or 3 ng / mL carrier, 4 ng / µL carrier or 4 ng / mL carrier, 5 ng / µL carrier or 5 ng / mL carrier, 6 ng / µL carrier or 6 ng / mL carrier, 7 ng / µL carrier or 7 ng / mL carrier, 8 ng / µL carrier or 8 ng / mL carrier, 9 ng / µL carrier or 9 ng / mL carrier, 10 ng / µL carrier or 10 ng / mL carrier, 11 ng / µL carrier or 11 ng / mL carrier, 12 ng / µL carrier or 13 ng / mL carrier. Carrier or 12ng / mL Carrier, 13ng / µL Carrier or 13ng / mL Carrier, 14ng / µL Carrier or 14ng / mL Carrier, 15ng / µL Carrier or 15ng / mL Carrier, 16ng / µL Carrier or 16ng / mL Carrier, 17ng / µL Carrier or 17ng / mL Carrier, 18ng / µL Carrier or 18ng / mL Carrier, 19ng / µL Carrier or 19ng / mL Carrier, 20ng / µL Carrier or 20ng / mL Carrier, 21ng / µL Carrier or 21ng / mL Carrier, 22ng / µL Carrier or 22ng / mL Carrier, 23ng / µL Carrier or 23ng / mL Carrier, 24ng / 24ng / µL carrier or 24ng / mL carrier, 25ng / µL carrier or 25ng / mL carrier, 26ng / µL carrier or 26ng / mL carrier, 27ng / µL carrier or 27ng / mL carrier, 28ng / µL carrier or 28ng / mL carrier, 29ng / µL carrier or 29ng / mL carrier, 30ng / µL carrier or 30ng / mL carrier, 31ng / µL carrier or 31ng / mL carrier, 32ng / µL carrier or 32ng / mL carrier, 33ng / µL carrier or 33ng / mL carrier, 34ng / µL carrier or 34ng / mL carrier, 35ng / µL carrier or 35ng / mL carrier, 36ng / µL carrier or 36ng / mL carrier, ng / µL Carrier or 36ng / mL Carrier, 37ng / µL Carrier or 37ng / mL Carrier, 38ng / µL Carrier or 38ng / mL Carrier, 39ng / µL Carrier or 39ng / mL Carrier, 40ng / µL Carrier or 40ng / mL Carrier, 41ng / µL Carrier or 41ng / mL Carrier, 42ng / µL Carrier or 42ng / mL Carrier, 43ng / µL Carrier or 43ng / mL Carrier, 44ng / µL Carrier or 44ng / mL Carrier, 45ng / µL Carrier or 45ng / mL Carrier, 46ng / µL Carrier or 46ng / mL Carrier, 47ng / µL Carrier or 47ng / mL Carrier,48ng / µL Vehicle or 48ng / mL Vehicle, 49ng / µL Vehicle or 49ng / mL Vehicle, 50ng / µL Vehicle or 50ng / mL Vehicle, 51ng / µL Vehicle or 51ng / mL Vehicle, 52ng / µL Vehicle or 52ng / mL Vehicle, 53ng / µL Vehicle or 53ng / mL Vehicle, 54ng / µL Vehicle or 54ng / mL Vehicle, 55ng / µL Vehicle or 55ng / mL Vehicle, 56ng / µL Vehicle or 56ng / mL Vehicle, 57ng / µL Vehicle or 57ng / mL Vehicle, 58ng / µL Vehicle or 58ng / mL Vehicle, 59ng / µL Vehicle or 59ng / mL Vehicle, 60ng / µL Vehicle or 60ng / mL carrier, 61ng / µL carrier or 61ng / mL carrier, 62ng / µL carrier or 62ng / mL carrier, 63ng / µL carrier or 63ng / mL carrier, 64ng / µL carrier or 64ng / mL carrier, 65ng / µL carrier or 65ng / mL carrier, 66ng / µL carrier or 66ng / mL carrier, 67ng / µL carrier or 67ng / mL carrier, 68ng / µL carrier or 68ng / mL carrier, 69ng / µL carrier or 69ng / mL carrier, 70ng / µL carrier or 70ng / mL carrier, 71ng / µL carrier or 71ng / mL carrier, 72ng / µL carrier or 72ng / mL carrier, 73ng / µL Vehicle or 73ng / mL Vehicle, 74ng / µL Vehicle or 74ng / mL Vehicle, 75ng / µL Vehicle or 75ng / mL Vehicle, 76ng / µL Vehicle or 76ng / mL Vehicle, 77ng / µL Vehicle or 77ng / mL Vehicle, 78ng / µL Vehicle or 78ng / mL Vehicle, 79ng / µL Vehicle or 79ng / mL Vehicle, 80ng / µL Vehicle or 80ng / mL Vehicle, 81ng / µL Vehicle or 81ng / mL Vehicle, 82ng / µL Vehicle or 82ng / mL Vehicle, 83ng / µL Vehicle or 83ng / mL Vehicle, 84ng / µL Vehicle or 84ng / mL Vehicle, 85ng / µL Vehicle or 85ng / mL carrier, 86ng / µL carrier or 86ng / mL carrier, 87ng / µL carrier or 87ng / mL carrier, 88ng / µL carrier or 88ng / mL carrier, 89ng / µL carrier or 89ng / mL carrier, 90ng / µL carrier or 90ng / mL carrier, 91ng / µL carrier or 91ng / mL carrier, 92ng / µL carrier or 92ng / mL carrier, 93ng / µL carrier or 93ng / mL carrier, 94ng / µL carrier or 94ng / mL carrier, 95ng / µL carrier or 95ng / mL carrier, 96ng / µL carrier or 96ng / mL carrier, 97ng / µL carrier or 97ng / mL carrier,98ng / µL carrier or 98ng / mL carrier, 99ng / µL carrier or 99ng / mL carrier, 100ng / µL carrier or 100ng / mL carrier, 101ng / µL carrier or 101ng / mL carrier, 102ng / µL carrier or 102ng / mL carrier, 103ng / µL carrier or 103ng / mL carrier, 104ng / µL carrier or 104ng / mL carrier, 105ng / µL carrier or 105ng / mL carrier, 106ng / µL carrier or 106ng / mL carrier, 107ng / µL carrier or 107ng / mL carrier, 108ng / µL carrier or 108ng / mL carrier, 109ng / µL carrier or 109ng / mL carrier Carrier or 109ng / mL Carrier, 110ng / µL Carrier or 110ng / mL Carrier, 111ng / µL Carrier or 111ng / mL Carrier, 112ng / µL Carrier or 112ng / mL Carrier, 113ng / µL Carrier or 113ng / mL Carrier, 114ng / µL Carrier or 114ng / mL Carrier, 115ng / µL Carrier or 115ng / mL Carrier, 116ng / µL Carrier or 116ng / mL Carrier, 117ng / µL Carrier or 117ng / mL Carrier, 118ng / µL Carrier or 118ng / mL Carrier, 119ng / µL Carrier or 119ng / mL Carrier, 120ng / µL Carrier or 12 0 ng / mL carrier, 121 ng / µL carrier or 121 ng / mL carrier, 122 ng / µL carrier or 122 ng / mL carrier, 123 ng / µL carrier or 123 ng / mL carrier, 124 ng / µL carrier or 124 ng / mL carrier, 125 ng / µL carrier or 125 ng / mL carrier, 126 ng / µL carrier or 126 ng / mL carrier, 127 ng / µL carrier or 127 ng / mL carrier, 128 ng / µL carrier or 128 ng / mL carrier, 129 ng / µL carrier or 129 ng / mL carrier, 130 ng / µL carrier or 130 ng / mL carrier, 131 ng / µL carrier or 131 ng / mL carrier mL carrier, 132ng / µL carrier or 132ng / mL carrier, 133ng / µL carrier or 133ng / mL carrier, 134ng / µL carrier or 134ng / mL carrier, 135ng / µL carrier or 135ng / mL carrier, 136ng / µL carrier or 136ng / mL carrier, 137ng / µL carrier or 137ng / mL carrier, 138ng / µL carrier or 138ng / mL carrier, 139ng / µL carrier or 139ng / mL carrier, 140ng / µL carrier or 140ng / mL carrier, 141ng / µL carrier or 141ng / mL carrier, 142ng / µL carrier or 142ng / mL carrier,143ng / µL carrier or 143ng / mL carrier, 144ng / µL carrier or 144ng / mL carrier, 145ng / µL carrier or 145ng / mL carrier, 146ng / µL carrier or 146ng / mL carrier, 147ng / µL carrier or 147ng / mL carrier, 148ng / µL carrier or 148ng / mL carrier, 149ng / µL carrier or 149ng / mL carrier, 150ng / µL carrier or 150ng / mL carrier, 151ng / µL carrier or 151ng / mL carrier, 152ng / µL carrier or 152ng / mL carrier, 153ng / µL carrier or 153ng / mL carrier, 154ng / µL Vehicle or 154ng / mL Vehicle, 155ng / µL Vehicle or 155ng / mL Vehicle, 156ng / µL Vehicle or 156ng / mL Vehicle, 157ng / µL Vehicle or 157ng / mL Vehicle, 158ng / µL Vehicle or 158ng / mL Vehicle, 159ng / µL Vehicle or 159ng / mL Vehicle, 160ng / µL Vehicle or 160ng / mL Vehicle, 161ng / µL Vehicle or 161ng / mL Vehicle, 162ng / µL Vehicle or 162ng / mL Vehicle, 163ng / µL Vehicle or 163ng / mL Vehicle, 164ng / µL Vehicle or 164ng / mL Vehicle, 165ng / µL Vehicle or 165ng / mL Vehicle, 166ng / µL Vehicle or 166ng / mL Vehicle, 167ng / µL Vehicle or 167ng / mL Vehicle, 168ng / µL Vehicle or 168ng / mL Vehicle, 169ng / µL Vehicle or 169ng / mL Vehicle, 170ng / µL Vehicle or 170ng / mL Vehicle, 171ng / µL Vehicle or 171ng / mL Vehicle, 172ng / µL Vehicle or 172ng / mL Vehicle, 173ng / µL Vehicle or 173ng / mL Vehicle, 174ng / µL Vehicle or 174ng / mL Vehicle, 175ng / µL Vehicle or 175ng / mL Vehicle, 176ng / µL Vehicle or 176ng / mL Carrier, 177ng / µL Carrier or 177ng / mL Carrier, 178ng / µL Carrier or 178ng / mL Carrier, 179ng / µL Carrier or 179ng / mL Carrier, 180ng / µL Carrier or 180ng / mL Carrier, 181ng / µL Carrier or 181ng / mL Carrier, 182ng / µL Carrier or 182ng / mL Carrier, 183ng / µL Carrier or 183ng / mL Carrier, 184ng / µL Carrier or 184ng / mL Carrier, 185ng / µL Carrier or 185ng / mL Carrier, 186ng / µL Carrier or 186ng / mL Carrier, 187ng / µL Carrier or 187ng / mL Carrier,188ng / µL carrier or 188ng / mL carrier, 189ng / µL carrier or 189ng / mL carrier, 190ng / µL carrier or 190ng / mL carrier, 191ng / µL carrier or 191ng / mL carrier, 192ng / µL carrier or 192ng / mL carrier, 193ng / µL carrier or 193ng / mL carrier, 194ng / µL carrier or 194ng / mL carrier, 195ng / µL carrier or 195ng / mL carrier, 196ng / µL carrier or 196ng / mL carrier, 197ng / µL carrier or 197ng / mL carrier, 198ng / µL carrier or 198ng / mL carrier, 199ng / µL of carrier or 199ng / mL of carrier, 200ng / µL of carrier or 200ng / mL of carrier, 201ng / µL of carrier or 201ng / mL of carrier, 202ng / µL of carrier or 202ng / mL of carrier, 203ng / µL of carrier or 203ng / mL of carrier, 204ng / µL of carrier or 204ng / mL of carrier, 205ng / µL of carrier or 205ng / mL of carrier, 206ng / µL of carrier or 206ng / mL of carrier, 207ng / µL of carrier or 207ng / mL of carrier, 208ng / µL of carrier or 208ng / mL of carrier, 209ng / µL of carrier or 209ng / mL of carrier, 210ng / µL of carrier or 211ng / mL of carrier 210ng / mL Vehicle, 211ng / µL Vehicle or 211ng / mL Vehicle, 212ng / µL Vehicle or 212ng / mL Vehicle, 213ng / µL Vehicle or 213ng / mL Vehicle, 214ng / µL Vehicle or 214ng / mL Vehicle, 215ng / µL Vehicle or 215ng / mL Vehicle, 216ng / µL Vehicle or 216ng / mL Vehicle, 217ng / µL Vehicle or 217ng / mL Vehicle, 218ng / µL Vehicle or 218ng / mL Vehicle, 219ng / µL Vehicle or 219ng / mL Vehicle, 220ng / µL Vehicle or 220ng / mL Vehicle, 221ng / µL Vehicle or 221ng / mL carrier, 222ng / µL carrier or 222ng / mL carrier, 223ng / µL carrier or 223ng / mL carrier, 224ng / µL carrier or 224ng / mL carrier, 225ng / µL carrier or 225ng / 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297ng / µL carrier or 297ng / mL carrier, 298ng / µL carrier or 298ng / mL carrier, 299ng / µL carrier or 299ng / mL carrier, 300ng / µL carrier or 300ng / µL carrier 300ng / mL carrier, 301ng / µL carrier or 301ng / mL carrier, 302ng / µL carrier or 302ng / mL carrier, 303ng / µL carrier or 303ng / mL carrier, 304ng / µL carrier or 304ng / mL carrier, 305ng / µL carrier or 305ng / mL carrier, 306ng / µL carrier or 306ng / mL carrier, 307ng / µL carrier or 307ng / mL carrier, 308ng / µL carrier or 308ng / mL carrier, 309ng / µL carrier or 309ng / mL carrier, 310ng / µL carrier or 310ng / mL carrier, 311ng / µL carrier or 311ng / mL carrier, 312ng / µL carrier or 312ng / mL carrier, 313ng / µL carrier or 313ng / mL carrier, 314ng / µL carrier or 314ng / mL carrier, 315ng / µL carrier or 315ng / mL carrier, 316ng / µL carrier or 316ng / mL carrier, 317ng / µL carrier or 317ng / mL carrier, 318ng / µL carrier or 318ng / mL carrier, 319ng / µL carrier or 319ng / mL carrier, 320ng / µL 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717ng / µL carrier or 717ng / mL carrier, 718ng / µL carrier or 718ng / mL carrier, 719ng / µL carrier or 719ng / mL carrier, 720ng / µL carrier or 720ng / mL carrier, 721ng / µL carrier or 721ng / mL carrier, 722ng / µL carrier or 722ng / mL carrier, 723ng / µL carrier or 723ng / mL carrier, 724ng / µL carrier or 724ng / mL carrier, 725ng / µL carrier or 725ng / mL carrier, 726ng / µL carrier or 726ng / mL carrier, 727ng / µL carrier or 727ng / mL carrier,728ng / µL carrier or 728ng / mL carrier, 729ng / µL carrier or 729ng / mL carrier, 730ng / µL carrier or 730ng / mL carrier, 731ng / µL carrier or 731ng / mL carrier, 732ng / µL carrier or 732ng / mL carrier, 733ng / µL carrier or 733ng / mL carrier, 734ng / µL carrier or 734ng / mL carrier, 735ng / µL carrier or 735ng / mL carrier, 736ng / µL carrier or 736ng / mL carrier, 737ng / µL carrier or 737ng / mL carrier, 738ng / µL carrier or 738ng / mL carrier, 739ng / µL of carrier or 739ng / mL of carrier, 740ng / µL of carrier or 740ng / mL of carrier, 741ng / µL of carrier or 741ng / mL of carrier, 742ng / µL of carrier or 742ng / mL of carrier, 743ng / µL of carrier or 743ng / mL of carrier, 744ng / µL of carrier or 744ng / mL of carrier, 745ng / µL of carrier or 745ng / mL of carrier, 746ng / µL of carrier or 746ng / mL of carrier, 747ng / µL of carrier or 747ng / mL of carrier, 748ng / µL of carrier or 748ng / mL of carrier, 749ng / µL of carrier or 749ng / mL of carrier, 750ng / µL of carrier or 750ng / mL carrier, 751ng / µL carrier or 751ng / mL carrier, 752ng / µL carrier or 752ng / mL carrier, 753ng / µL carrier or 753ng / mL carrier, 754ng / µL carrier or 754ng / mL carrier, 755ng / µL carrier or 755ng / mL carrier, 756ng / µL carrier or 756ng / mL carrier, 757ng / µL carrier or 757ng / mL carrier, 758ng / µL carrier or 758ng / mL carrier, 759ng / µL carrier or 759ng / mL carrier, 760ng / µL carrier or 760ng / mL carrier, 761ng / µL carrier or 761ng / mL carrier, 762ng / µL carrier or 762ng / mL carrier, 763ng / µL carrier or 763ng / mL carrier, 764ng / µL carrier or 764ng / mL carrier, 765ng / µL carrier or 765ng / mL carrier, 766ng / µL carrier or 766ng / mL carrier, 767ng / µL carrier or 767ng / mL carrier, 768ng / µL carrier or 768ng / mL carrier, 769ng / µL carrier or 769ng / mL carrier, 770ng / µL carrier or 770ng / mL carrier, 771ng / µL carrier or 771ng / mL carrier, 772ng / µL carrier or 772ng / mL carrier,773ng / µL carrier or 773ng / mL carrier, 774ng / µL carrier or 774ng / mL carrier, 775ng / µL carrier or 775ng / mL carrier, 776ng / µL carrier or 776ng / mL carrier, 777ng / µL carrier or 777ng / mL carrier, 778ng / µL carrier or 778ng / mL carrier, 779ng / µL carrier or 779ng / mL carrier, 780ng / µL carrier or 780ng / mL carrier, 781ng / µL carrier or 781ng / mL carrier, 782ng / µL carrier or 782ng / mL carrier, 783ng / µL carrier or 783ng / mL carrier, 784ng / µL of carrier or 784ng / mL of carrier, 785ng / µL of carrier or 785ng / mL of carrier, 786ng / µL of carrier or 786ng / mL of carrier, 787ng / µL of carrier or 787ng / mL of carrier, 788ng / µL of carrier or 788ng / mL of carrier, 789ng / µL of carrier or 789ng / mL of carrier, 790ng / µL of carrier or 790ng / mL of carrier, 791ng / µL of carrier or 791ng / mL of carrier, 792ng / µL of carrier or 792ng / mL of carrier, 793ng / µL of carrier or 793ng / mL of carrier, 794ng / µL of carrier or 794ng / mL of carrier, 795ng / µL of carrier or 796ng / mL of carrier 795ng / mL carrier, 796ng / µL carrier or 796ng / mL carrier, 797ng / µL carrier or 797ng / mL carrier, 798ng / µL carrier or 798ng / mL carrier, 799ng / µL carrier or 799ng / mL carrier, 800ng / µL carrier or 800ng / mL carrier, 801ng / µL carrier or 801ng / mL carrier, 802ng / µL carrier or 802ng / mL carrier, 803ng / µL carrier or 803ng / mL carrier, 804ng / µL carrier or 804ng / mL carrier, 805ng / µL carrier or 805ng / mL carrier, 806ng / µL carrier or 806ng / mL carrier, 807ng / µL carrier or 807ng / mL carrier, 808ng / µL carrier or 808ng / mL carrier, 809ng / µL carrier or 809ng / mL carrier, 810ng / µL carrier or 810ng / mL carrier, 811ng / µL carrier or 811ng / mL carrier, 812ng / µL carrier or 812ng / mL carrier, 813ng / µL carrier or 813ng / mL carrier, 814ng / µL carrier or 814ng / mL carrier, 815ng / µL carrier or 815ng / mL carrier, 816ng / µL carrier or 816ng / mL carrier, 817ng / µL carrier or 817ng / mL carrier,818ng / µL carrier or 818ng / mL carrier, 819ng / µL carrier or 819ng / mL carrier, 820ng / µL carrier or 820ng / mL carrier, 821ng / µL carrier or 821ng / mL carrier, 822ng / µL carrier or 822ng / mL carrier, 823ng / µL carrier or 823ng / mL carrier, 824ng / µL carrier or 824ng / mL carrier, 825ng / µL carrier or 825ng / mL carrier, 826ng / µL carrier or 826ng / mL carrier, 827ng / µL carrier or 827ng / mL carrier, 828ng / µL carrier or 828ng / mL carrier, 829ng / µL carrier or 829ng / mL carrier, 830ng / µL carrier or 830ng / mL carrier, 831ng / µL carrier or 831ng / mL carrier, 832ng / µL carrier or 832ng / mL carrier, 833ng / µL carrier or 833ng / mL carrier, 834ng / µL carrier or 834ng / mL carrier, 835ng / µL carrier or 835ng / mL carrier, 836ng / µL carrier or 836ng / mL carrier, 837ng / µL carrier or 837ng / mL carrier, 838ng / µL carrier or 838ng / mL carrier, 839ng / µL carrier or 839ng / mL carrier, 840ng / µL carrier or 840ng / mL carrier, 841ng / µL carrier or 841ng / mL carrier, 842ng / µL carrier or 842ng / mL carrier, 843ng / µL carrier or 843ng / mL carrier, 844ng / µL carrier or 844ng / mL carrier, 845ng / µL carrier or 845ng / mL carrier, 846ng / µL carrier or 846ng / mL carrier, 847ng / µL carrier or 847ng / mL carrier, 848ng / µL carrier or 848ng / mL carrier, 849ng / µL carrier or 849ng / mL carrier, 850ng / µL carrier or 850ng / mL carrier, 851ng / µL carrier or 851ng / mL carrier, 852ng / µL carrier or 852ng / mL carrier, 853ng / µL carrier or 853ng / mL carrier, 854ng / µL carrier or 854ng / mL carrier, 855ng / µL carrier or 855ng / mL carrier, 856ng / µL carrier or 856ng / mL carrier, 857ng / µL carrier or 857ng / mL carrier, 858ng / µL carrier or 858ng / mL carrier, 859ng / µL carrier or 859ng / mL carrier, 860ng / µL carrier or 860ng / mL carrier, 861ng / µL carrier or 861ng / mL carrier, 862ng / µL carrier or 862ng / mL carrier,863ng / µL carrier or 863ng / mL carrier, 864ng / µL carrier or 864ng / mL carrier, 865ng / µL carrier or 865ng / mL carrier, 866ng / µL carrier or 866ng / mL carrier, 867ng / µL carrier or 867ng / mL carrier, 868ng / µL carrier or 868ng / mL carrier, 869ng / µL carrier or 869ng / mL carrier, 870ng / µL carrier or 870ng / mL carrier, 871ng / µL carrier or 871ng / mL carrier, 872ng / µL carrier or 872ng / mL carrier, 873ng / µL carrier or 873ng / mL carrier, 874ng / µL of carrier or 874ng / mL of carrier, 875ng / µL of carrier or 875ng / mL of carrier, 876ng / µL of carrier or 876ng / mL of carrier, 877ng / µL of carrier or 877ng / mL of carrier, 878ng / µL of carrier or 878ng / mL of carrier, 879ng / µL of carrier or 879ng / mL of carrier, 880ng / µL of carrier or 880ng / mL of carrier, 881ng / µL of carrier or 881ng / mL of carrier, 882ng / µL of carrier or 882ng / mL of carrier, 883ng / µL of carrier or 883ng / mL of carrier, 884ng / µL of carrier or 884ng / mL of carrier, 885ng / µL of carrier or 885ng / mL carrier, 886ng / µL carrier or 886ng / mL carrier, 887ng / µL carrier or 887ng / mL carrier, 888ng / µL carrier or 888ng / mL carrier, 889ng / µL carrier or 889ng / mL carrier, 890ng / µL carrier or 890ng / mL carrier, 891ng / µL carrier or 891ng / mL carrier, 892ng / µL carrier or 892ng / mL carrier, 893ng / µL carrier or 893ng / mL carrier, 894ng / µL carrier or 894ng / mL carrier, 895ng / µL carrier or 895ng / mL carrier, 896ng / µL carrier or 896ng / mL carrier, 897ng / µL carrier or 897ng / mL carrier, 898ng / µL carrier or 898ng / mL carrier, 899ng / µL carrier or 899ng / mL carrier, 900ng / µL carrier or 900ng / mL carrier, 901ng / µL carrier or 901ng / mL carrier, 902ng / µL carrier or 902ng / mL carrier, 903ng / µL carrier or 903ng / mL carrier, 904ng / µL carrier or 904ng / mL carrier, 905ng / µL carrier or 905ng / mL carrier, 906ng / µL carrier or 906ng / mL carrier, 907ng / µL carrier or 907ng / mL carrier,908ng / µL carrier or 908ng / mL carrier, 909ng / µL carrier or 909ng / mL carrier, 910ng / µL carrier or 910ng / mL carrier, 911ng / µL carrier or 911ng / mL carrier, 912ng / µL carrier or 912ng / mL carrier, 913ng / µL carrier or 913ng / mL carrier, 914ng / µL carrier or 914ng / mL carrier, 915ng / µL carrier or 915ng / mL carrier, 916ng / µL carrier or 916ng / mL carrier, 917ng / µL carrier or 917ng / mL carrier, 918ng / µL carrier or 918ng / mL carrier, 919ng / µL carrier or 919ng / mL carrier, 920ng / µL carrier or 920ng / mL carrier, 921ng / µL carrier or 921ng / mL carrier, 922ng / µL carrier or 922ng / mL carrier, 923ng / µL carrier or 923ng / mL carrier, 924ng / µL carrier or 924ng / mL carrier, 925ng / µL carrier or 925ng / mL carrier, 926ng / µL carrier or 926ng / mL carrier, 927ng / µL carrier or 927ng / mL carrier, 928ng / µL carrier or 928ng / mL carrier, 929ng / µL carrier or 929ng / mL carrier, 930ng / µL carrier or 930ng / mL carrier, 931ng / µL carrier or 931ng / mL carrier, 932ng / µL carrier or 932ng / mL carrier, 933ng / µL carrier or 933ng / mL carrier, 934ng / µL carrier or 934ng / mL carrier, 935ng / µL carrier or 935ng / mL carrier, 936ng / µL carrier or 936ng / mL carrier, 937ng / µL carrier or 937ng / mL carrier, 938ng / µL carrier or 938ng / mL carrier, 939ng / µL carrier or 939ng / mL carrier, 940ng / µL carrier or 940ng / mL carrier, 941ng / µL carrier or 941ng / mL carrier, 942ng / µL carrier or 942ng / mL carrier, 943ng / µL carrier or 943ng / mL carrier, 944ng / µL carrier or 944ng / mL carrier, 945ng / µL carrier or 945ng / mL carrier, 946ng / µL carrier or 946ng / mL carrier, 947ng / µL carrier or 947ng / mL carrier, 948ng / µL carrier or 948ng / mL carrier, 949ng / µL carrier or 949ng / mL carrier, 950ng / µL carrier or 950ng / mL carrier, 951ng / µL carrier or 951ng / mL carrier, 952ng / µL carrier or 952ng / mL carrier,953ng / µL carrier or 953ng / mL carrier, 954ng / µL carrier or 954ng / mL carrier, 955ng / µL carrier or 955ng / mL carrier, 956ng / µL carrier or 956ng / mL carrier, 957ng / µL carrier or 957ng / mL carrier, 958ng / µL carrier or 958ng / mL carrier, 959ng / µL carrier or 959ng / mL carrier, 960ng / µL carrier or 960ng / mL carrier, 961ng / µL carrier or 961ng / mL carrier, 962ng / µL carrier or 962ng / mL carrier, 963ng / µL carrier or 963ng / mL carrier, 964ng / µL carrier or 964ng / mL carrier, 965ng / µL carrier or 965ng / mL carrier, 966ng / µL carrier or 966ng / mL carrier, 967ng / µL carrier or 967ng / mL carrier, 968ng / µL carrier or 968ng / mL carrier, 969ng / µL carrier or 969ng / mL carrier, 970ng / µL carrier or 970ng / mL carrier, 971ng / µL carrier or 971ng / mL carrier, 972ng / µL carrier or 972ng / mL carrier, 973ng / µL carrier or 973ng / mL carrier, 974ng / µL carrier or 974ng / mL carrier, 975ng / µL carrier or 975ng / mL carrier, 976ng / µL carrier or 976ng / mL carrier, 977ng / µL carrier or 977ng / mL carrier, 978ng / µL carrier or 978ng / mL carrier, 979ng / µL carrier or 979ng / mL carrier, 980ng / µL carrier or 980ng / mL carrier, 981ng / µL carrier or 981ng / mL carrier, 982ng / µL carrier or 982ng / mL carrier, 983ng / µL carrier or 983ng / mL carrier, 984ng / µL carrier or 984ng / mL carrier, 985ng / µL carrier or 985ng / mL carrier, 986ng / µL carrier or 986ng / mL carrier, 987ng / µL carrier or 987ng / mL carrier, 988ng / µL carrier or 988ng / mL carrier, 989ng / µL carrier or 989ng / mL carrier, 990ng / µL carrier or 990ng / mL carrier, 991ng / µL carrier or 991ng / mL carrier, 992ng / µL carrier or 992ng / mL carrier, 993ng / µL carrier or 993ng / mL carrier, 994ng / µL carrier or 994ng / mL carrier, 995ng / µL carrier or 995ng / mL carrier, 996ng / µL carrier or 996ng / mL carrier, 997ng / µL carrier or 997ng / mL carrier,998 ng / µL carrier or 998 ng / mL carrier, 999 ng / µL carrier or 999 ng / mL carrier, 1000 ng / µL carrier or 1000 ng / mL carrier (or higher than 1000 ng / µL carrier or 1000 ng / mL carrier or any range derivable therefrom). The carrier can be any carrier suitable for contacting proteins and / or nucleic acids, such as water (including nuclease-free water), a buffered solution, a saline solution, DMSO, or other suitable liquid. The carrier can be a cell culture medium. The cells can be provided with or contacted with an effective amount of the solution. The effective amount of the solution can be determined by one skilled in the art and can be 1 pL, nL or µL, 2 pL, nL or µL, 3 pL, nL or µL, 4 pL, nL or µL, 5 pL, nL or µL, 6 pL, nL or µL, 7 pL, nL or µL, 8 pL, nL or µL, 9 pL, nL or µL, 10 pL, nL or µL, 11 pL, nL or µL, 12 pL, nL or µL, 13 pL, nL or µL, 14 pL, nL or µL, 15 pL, nL or µL, 16 pL, nL or µL, 17 pL, nL or µL, 18pL, nL, or µL, 19pL, nL, or µL, 20pL, nL, or µL, 21pL, nL, or µL, 22pL, nL, or µL, 23pL, nL, or µL, 24pL, nL, or µL, 25pL, nL, or µL, 26pL, nL, or µL, 27pL, nL, or µL, 28pL, nL, or µL, 29pL, nL, or µL, 30pL, nL, or µL, 31pL, nL, or µL, 32pL, nL, or µL, 33pL, nL, or µL, 34pL, nL, or µL, 35pL, nL, or µL, 36 pL, nL, or µL, 37pL, nL, or µL, 38pL, nL, or µL, 39pL, nL, or µL, 40pL, nL, or µL, 41pL, nL, or µL, 42pL, nL, or µL, 43pL, nL, or µL, 44pL, nL, or µL, 45pL, nL, or µL, 46pL, nL, or µL, 47pL, nL, or µL, 48pL, nL, or µL, 49pL, nL, or µL, 50pL, nL, or µL, 51pL, nL, or µL, 52pL, nL, or µL, 53pL, nL, or µL, 54p L, nL, or µL, 55pL, nL, or µL, 56pL, nL, or µL, 57pL, nL, or µL, 58pL, nL, or µL, 59pL, nL, or µL, 60pL, nL, or µL, 61pL, nL, or µL, 62pL, nL, or µL, 63pL, nL, or µL, 64pL, nL, or µL, 65pL, nL, or µL, 66pL, nL, or µL, 67pL, nL, or µL, 68pL, nL, or µL, 69pL, nL, or µL, 70pL, nL, or µL, 71pL, nL, or µL, 72pL,nL or µL, 73pL, nL or µL, 74pL, nL or µL, 75pL, nL or µL, 76pL, nL or µL, 77pL, nL or µL, 78pL, nL or µL, 79pL, nL or µL, 80pL, nL or µL, 81pL, nL or µL, 82pL, nL or µL, 83pL, nL or µL, 84pL, nL, or µL, 85pL, nL, or µL, 86pL, nL, or µL, 87pL, nL, or µL, 88pL, nL, or µL, 89pL, nL, or µL, 90pL, nL, or µL, 91pL, nL, or µL, 92pL, nL, or µL, 93pL, nL, or µL, 94pL, nL, or µL, 95pL, nL, or µL, 96pL, nL, or µL, 97pL, nL, or µL, 98pL, nL, or µL, 99pL, nL, or µL, 100pL, nL, or µL, 101pL, nL, or µL, 102pL, nL, or µL, 103pL, nL, or µL, 104pL, nL, or µL, 105pL, nL, or µL, 106pL, nL, or µL, 107pL, nL, or µL, 108pL, nL, or µL, 109pL, nL, or µL, 110pL, nL, or µL, 111pL, nL, or µL, 112pL, nL, or µL, 113pL, nL, or µL, 114pL, nL, or µL, 115pL, nL, or µL, 1 16pL, nL, or µL, 117pL, nL, or µL, 118pL, nL, or µL, 119pL, nL, or µL, 120pL, nL, or µL, 121pL, nL, or µL, 122pL, nL, or µL, 123pL, nL, or µL, 124pL, nL, or µL, 125pL, nL, or µL, 126pL , nL or µL, 127pL, nL or µL, 128pL, nL or µL, 129pL, nL or µL, 130pL, nL or µL, 131pL, nL or µL, 132pL, nL or µL, 133pL, nL or µL, 134pL, nL or µL, 135pL, nL or µL, 136pL, nL or µL, 137pL, nL, or µL, 138pL, nL, or µL, 139pL, nL, or µL, 140pL, nL, or µL, 141pL, nL, or µL, 142pL, nL, or µL, 143pL, nL, or µL, 144pL, nL, or µL, 145pL, nL, or µL, 146pL, nL, or µL, 1 47pL, nL, or µL, 148pL, nL, or µL, 149pL, nL, or µL, 150pL, nL, or µL, 151pL, nL, or µL, 152pL, nL, or µL, 153pL, nL, or µL, 154pL, nL, or µL, 155pL, nL, or µL, 156pL, nL, or µL, 157pL,nL or µL, 158pL, nL or µL, 159pL, nL or µL, 160pL, nL or µL, 161pL, nL or µL, 162pL, nL or µL, 163pL, nL or µL, 164pL, nL or µL, 165pL, nL or µL, 166pL, nL or µL, 167pL, nL or µ L, 168pL, nL, or µL, 169pL, nL, or µL, 170pL, nL, or µL, 171pL, nL, or µL, 172pL, nL, or µL, 173pL, nL, or µL, 174pL, nL, or µL, 175pL, nL, or µL, 176pL, nL, or µL, 177pL, nL, or µL, 178 pL, nL, or µL, 179pL, nL, or µL, 180pL, nL, or µL, 181pL, nL, or µL, 182pL, nL, or µL, 183pL, nL, or µL, 184pL, nL, or µL, 185pL, nL, or µL, 186pL, nL, or µL, 187pL, nL, or µL, 188pL, nL L or µL, 189pL, nL or µL, 190pL, nL or µL, 191pL, nL or µL, 192pL, nL or µL, 193pL, nL or µL, 194pL, nL or µL, 195pL, nL or µL, 196pL, nL or µL, 197pL, nL or µL, 198pL, nL or µL, 199pL, nL, or µL, 200pL, nL, or µL, 201pL, nL, or µL, 202pL, nL, or µL, 203pL, nL, or µL, 204pL, nL, or µL, 205pL, nL, or µL, 206pL, nL, or µL, 207pL, nL, or µL, 208pL, nL, or µL, 209pL L, nL, or µL, 210pL, nL, or µL, 211pL, nL, or µL, 212pL, nL, or µL, 213pL, nL, or µL, 214pL, nL, or µL, 215pL, nL, or µL, 216pL, nL, or µL, 217pL, nL, or µL, 218pL, nL, or µL, 219pL, nL, or µL, 220pL, nL, or µL, 221pL, nL, or µL, 222pL, nL, or µL, 223pL, nL, or µL, 224pL, nL, or µL, 225pL, nL, or µL, 226pL, nL, or µL, 227pL, nL, or µL, 228pL, nL, or µL, 229pL, nL, or µL, 2 30pL, nL, or µL, 231pL, nL, or µL, 232pL, nL, or µL, 233pL, nL, or µL, 234pL, nL, or µL, 235pL, nL, or µL, 236pL, nL, or µL, 237pL, nL, or µL, 238pL, nL, or µL, 239pL, nL, or µL, 240pL,nL or µL, 241pL, nL or µL, 242pL, nL or µL, 243pL, nL or µL, 244pL, nL or µL, 245pL, nL or µL, 246pL, nL or µL, 247pL, nL or µL, 248pL, nL or µL, 249pL, nL or µL, 250pL, nL or µ L, 251pL, nL, or µL, 252pL, nL, or µL, 253pL, nL, or µL, 254pL, nL, or µL, 255pL, nL, or µL, 256pL, nL, or µL, 257pL, nL, or µL, 258pL, nL, or µL, 259pL, nL, or µL, 260pL, nL, or µL, 261 pL, nL, or µL, 262pL, nL, or µL, 263pL, nL, or µL, 264pL, nL, or µL, 265pL, nL, or µL, 266pL, nL, or µL, 267pL, nL, or µL, 268pL, nL, or µL, 269pL, nL, or µL, 270pL, nL, or µL, 271pL, nL L or µL, 272pL, nL or µL, 273pL, nL or µL, 274pL, nL or µL, 275pL, nL or µL, 276pL, nL or µL, 277pL, nL or µL, 278pL, nL or µL, 279pL, nL or µL, 280pL, nL or µL, 281pL, nL or µL, 282pL, nL, or µL, 283pL, nL, or µL, 284pL, nL, or µL, 285pL, nL, or µL, 286pL, nL, or µL, 287pL, nL, or µL, 288pL, nL, or µL, 289pL, nL, or µL, 290pL, nL, or µL, 291pL, nL, or µL, 292p L, nL, or µL, 293pL, nL, or µL, 294pL, nL, or µL, 295pL, nL, or µL, 296pL, nL, or µL, 297pL, nL, or µL, 298pL, nL, or µL, 299pL, nL, or µL, 300pL, nL, or µL, 301pL, nL, or µL, 302pL, nL, or µL, 303pL, nL, or µL, 304pL, nL, or µL, 305pL, nL, or µL, 306pL, nL, or µL, 307pL, nL, or µL, 308pL, nL, or µL, 309pL, nL, or µL, 310pL, nL, or µL, 311pL, nL, or µL, 312pL, nL, or µL, 3 13pL, nL, or µL, 314pL, nL, or µL, 315pL, nL, or µL, 316pL, nL, or µL, 317pL, nL, or µL, 318pL, nL, or µL, 319pL, nL, or µL, 320pL, nL, or µL, 321pL, nL, or µL, 322pL, nL, or µL, 323pL,nL or µL, 324pL, nL or µL, 325pL, nL or µL, 326pL, nL or µL, 327pL, nL or µL, 328pL, nL or µL, 329pL, nL or µL, 330pL, nL or µL, 331pL, nL or µL, 332pL, nL or µL, 333pL, nL or µ L, 334pL, nL, or µL, 335pL, nL, or µL, 336pL, nL, or µL, 337pL, nL, or µL, 338pL, nL, or µL, 339pL, nL, or µL, 340pL, nL, or µL, 341pL, nL, or µL, 342pL, nL, or µL, 343pL, nL, or µL, 344 pL, nL, or µL, 345pL, nL, or µL, 346pL, nL, or µL, 347pL, nL, or µL, 348pL, nL, or µL, 349pL, nL, or µL, 350pL, nL, or µL, 351pL, nL, or µL, 352pL, nL, or µL, 353pL, nL, or µL, 354pL, nL L or µL, 355pL, nL or µL, 356pL, nL or µL, 357pL, nL or µL, 358pL, nL or µL, 359pL, nL or µL, 360pL, nL or µL, 361pL, nL or µL, 362pL, nL or µL, 363pL, nL or µL, 364pL, nL or µL, 365pL, nL, or µL, 366pL, nL, or µL, 367pL, nL, or µL, 368pL, nL, or µL, 369pL, nL, or µL, 370pL, nL, or µL, 371pL, nL, or µL, 372pL, nL, or µL, 373pL, nL, or µL, 374pL, nL, or µL, 375p L, nL, or µL, 376pL, nL, or µL, 377pL, nL, or µL, 378pL, nL, or µL, 379pL, nL, or µL, 380pL, nL, or µL, 381pL, nL, or µL, 382pL, nL, or µL, 383pL, nL, or µL, 384pL, nL, or µL, 385pL, nL, or µL, 386pL, nL, or µL, 387pL, nL, or µL, 388pL, nL, or µL, 389pL, nL, or µL, 390pL, nL, or µL, 391pL, nL, or µL, 392pL, nL, or µL, 393pL, nL, or µL, 394pL, 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or µL, 844pL, nL, or µL, 845pL, nL, or µL, 846pL, nL, or µL, 847pL, nL, or µL, 848pL, nL, or µL, 849pL, nL, or µL, 850pL, nL, or µL, 851pL, nL, or µL, 852pL, nL L or µL, 853pL, nL or µL, 854pL, nL or µL, 855pL, nL or µL, 856pL, nL or µL, 857pL, nL or µL, 858pL, nL or µL, 859pL, nL or µL, 860pL, nL or µL, 861pL, nL or µL, 862pL, nL or µL, 863pL, nL, or µL, 864pL, nL, or µL, 865pL, nL, or µL, 866pL, nL, or µL, 867pL, nL, or µL, 868pL, nL, or µL, 869pL, nL, or µL, 870pL, nL, or µL, 871pL, nL, or µL, 872pL, nL, or µL, 873p L, nL, or µL, 874pL, nL, or µL, 875pL, nL, or µL, 876pL, nL, or µL, 877pL, nL, or µL, 878pL, nL, or µL, 879pL, nL, or µL, 880pL, nL, or µL, 881pL, nL, or µL, 882pL, nL, or µL, 883pL, nL, or µL, 884pL, nL, or µL, 885pL, nL, or µL, 886pL, nL, or µL, 887pL, nL, or µL, 888pL, nL, or µL, 889pL, nL, or µL, 890pL, nL, or µL, 891pL, nL, or µL, 892pL, nL, or µL, 893pL, nL, or µL, 8 94pL, nL, or µL, 895pL, nL, or µL, 896pL, nL, or µL, 897pL, nL, or µL, 898pL, nL, or µL, 899pL, nL, 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L, nL, or µL, 957pL, nL, or µL, 958pL, nL, or µL, 959pL, nL, or µL, 960pL, nL, or µL, 961pL, nL, or µL, 962pL, nL, or µL, 963pL, nL, or µL, 964pL, nL, or µL, 965pL, nL, or µL, 966pL, nL, or µL, 967pL, nL, or µL, 968pL, nL, or µL, 969pL, nL, or µL, 970pL, nL, or µL, 971pL, nL, or µL, 972pL, nL, or µL, 973pL, nL, or µL, 974pL, nL, or µL, 975pL, nL, or µL, 976pL, nL, or µL, 9 77pL, nL, or µL, 978pL, nL, or µL, 979pL, nL, or µL, 980pL, nL, or µL, 981pL, nL, or µL, 982pL, nL, or µL, 983pL, nL, or µL, 984pL, nL, or µL, 985pL, nL, or µL, 986pL, nL, or µL, 987pL,nL or µL, 988pL, nL or µL, 989pL, nL or µL, 990pL, nL or µL, 991pL, nL or µL, 992pL, nL or µL, 993pL, nL or µL, 994pL, nL or µL, 995pL, nL or µL, 996pL, nL or µL, 997pL, nL or µL, 998pL, nL or µL, 999pL, nL or µL, 1000pL, nL or µL, or any range derivative therefrom.

[0019] Also disclosed are aspects in which FTO is replaced by different RNA demethylases or additionally uses different RNA demethylases. In some aspects, the different RNA demethylases are ALKBH5. In some aspects, the different RNA demethylases can be used in the same manner as FTO, including administering in the same manner. Also disclosed are nucleic acid molecules encoding different RNA demethylases.

[0020] Nucleic acids and / or proteins can be provided in a vector capable of introducing the nucleic acid and / or protein into cells, such as liposomes or viruses. Nucleic acids and / or proteins can be introduced into cells by injection of the nucleic acid and / or protein into the cells, including by microinjection, in a volume of less than 1 µL. Nucleic acids can be introduced via viral vectors and / or by transfection. Introduction can be performed by contacting cells with the protein and / or nucleic acid.

[0021] The amount of exogenous FTO protein present in fertilized cells due to contact of the cells with FTO protein and / or FTO nucleic acid can decrease in each cell as the fertilized cells expand and divide. In some aspects, when the fertilized cells are in the embryonic development stage, exogenous FTO cannot be detected in the fertilized cells.

[0022] Fertilized cells can be fertilized by any method, including in vitro fertilization. Fertilized cells can also be produced by natural mating, artificial insemination, multiple ovulation and embryo transfer, somatic cell nuclear transfer, or other methods of fertilizing cells. Fertilized cells can be collected by non-surgical or surgical retrieval techniques.

[0023] The animal can be any animal, including humans, non-human primates, livestock animals, companion animals, rodents, or endangered animals. The animal can be transgenic. Livestock animals can be ungulates, such as cattle, pigs, sheep, bison, or deer. Livestock animals can be birds, including any poultry, such as chickens, ducks, geese, pigeons, or turkeys. Livestock animals can be aquatic animals, such as fish or shellfish. Livestock can be animals raised for food. Companion animals can be any domesticated animals, such as dogs, cats, horses, or rabbits. Rodents can be any rodents, including those used in biomedical research, such as mice or rats. Endangered animals can be any threatened or endangered wild animal, such as pandas, bison, polar bears, tigers, or lions. Endangered animals may be classified as endangered species. It is particularly contemplated that any specific animal identified herein may be excluded in one or more aspects.

[0024] In certain aspects, fertilized cells are xenotransplanted into an animal. For example, fertilized cells can be from one species but implanted into an animal of another species. The animal can be used for medical xenotransplantation products, such as a pig. Such an animal can be used for xenotransplantation products.

[0025] In certain aspects, the nucleic acid molecule encoding FTO is a DNA molecule, an RNA molecule, or any nucleic acid analog molecule. The nucleic acid molecule encoding FTO can be an mRNA molecule. The DNA molecule can be an expression construct, including any expression construct capable of transiently overexpressing FTO protein.

[0026] In some aspects, nucleic acid is provided to cells or contacted with cells in the presence or absence of other solutes, carriers, activators or other components used by those skilled in the art for implementing the methods described herein. In some aspects, cells including fertilized cells are implanted in the presence or absence of other solutes, carriers, activators or other components used by those skilled in the art for implementing the methods described herein.

[0027] Also disclosed are cells containing exogenous FTO protein and / or nucleic acid encoding exogenous FTO protein. Any method can be used to generate cells, including any method described herein for introducing FTO protein and / or FTO nucleic acid into cells. The cells can be fertilized cells, germ cells, or gametes.

[0028] Also disclosed are compositions comprising any of the cells disclosed herein.The composition may also comprise one or more than one reagent for in vitro fertilization.

[0029] Throughout this application, the term "about" is used according to its ordinary and customary meaning in the arts of cell and molecular biology to indicate a value that includes the standard deviation of error for the device or method being employed to determine the value.

[0030] When used in conjunction with the terms "including" or "comprising", the use of the word "a" or "an" can mean "one", but it can also be consistent with the meaning of "one or more" or "one or more", "at least one" or "at least one", and "one or more than one" or "one or more than one". Plural references are not used.

[0031] As used herein, the terms "or" and "and / or" are used to describe combinations or mutual exclusions of multiple components. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z can be specifically excluded from one or more aspects.

[0032] The words "comprising," "including," "having," "characterized by," "containing," and their similar expressions are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0033] The compositions and methods of use thereof can "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed throughout this specification. The phrase "consisting of" excludes any unspecified element, step, or ingredient. The phrase "consisting essentially of" limits the scope of the subject matter to the specified materials or steps, and those that do not materially affect the basic and novel characteristics thereof. It is contemplated that one or more aspects described in the context of the term "comprising" or "including" can also be implemented in the context of the term "consisting of" or "consisting essentially of."

[0034] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Additionally, the compositions of the invention can be used to implement the methods of the invention.

[0035] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect can also be described in the language of a "use" claim, such as "use" of any compound, composition, or agent discussed herein to achieve or carry out the therapeutic, diagnostic, or physiological purpose or effect.

[0036] The purposes of one or more than one sequence or composition can be applied based on any method described herein. Other one or more than one aspects are discussed in the full text of this application. Any one or more than one aspect discussed about one aspect of the present disclosure is also applicable to other aspects of the present disclosure, and vice versa.

[0037] It is specifically contemplated that any limitation discussed with respect to one or more aspects of the present invention may apply to any other one or more aspects of the present invention. Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to produce or use any composition of the present invention. Aspects of the aspects described in the Examples are also aspects that may be implemented elsewhere in the context of aspects discussed in various Examples or elsewhere in this application, such as in the Summary of the Invention, Detailed Description of the Invention, Claims, and Description of the Figures.

[0038] It is also specifically contemplated that certain limitations that are not specifically disclosed as not existing in the clearly defined systems, compositions, and / or methods, but are understood by one of ordinary skill in the art to be necessarily absent, may be excluded when practicing the aspects disclosed herein. For example, limitations that are not specifically disclosed as not existing but are required to exist in order for the disclosed systems, compositions, and / or methods to be inventive relative to the prior art may be excluded. Such limitations include aspects that are currently standard when performing similar methods and / or making or using similar systems or compositions.

[0039] Other objects, features and advantages of the present invention will become readily apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific aspects of the present invention, are given only as illustrations, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following drawings form part of this specification and are included to further illustrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific aspects provided herein.

[0041] Figure 1 A to Figure 1 E. In mESCs, mRNA expression of LINE1 RNA 6 A is the main substrate of FTO. (1A) shows the hypermethylated mRNA on carRNA after FTO KO. 6 m of peak A 6 A. Violin plot of horizontal fold change. P values were determined using the Wilcoxon rank sum test. (1B) shows the difference between the two groups. 6 Box plot of the fold change in expression of A-labeled carRNA compared to hypermethylated carRNA after Fto KO. P values were determined using the Wilcoxon signed-rank test. (1C) Overview of repetitive RNAs on chromatin after Fto KO. Top: Number of hypermethylated peaks, average m 6A level and expression. 6 A: Fold changes in expression levels and expression levels (p values determined using the Wilcoxon signed rank test). Bottom: Fold changes in expression levels of hypermethylated and non-hypermethylated repetitive RNAs (p values determined using the Wilcoxon rank sum test). (1D) shows the mRNA expression of LINE1 RNA after Fto KO. 6 A scatter plot of the negative correlation between the fold change of the level of expression and the fold change of the expression level. 6 Fold changes in A levels, LINE1 RNA was divided into 100 bins. r refers to the Pearson correlation coefficient. P values were calculated based on the t-distribution. (1E) GSEA from mRNA-seq showing upregulated 2C genes after Fto KO (top) and downregulated ESC-highly expressed genes (bottom). NES, normalized enrichment score.

[0042] Figure 2 A to Figure 2 F. FTO by m 6 A demethylation regulates LINE1 RNA levels. (2A) WT mESC and Fto - / - mESC 6 m on A-labeled LINE1 RNA 6 Peak A spectrum. (2B) shows the m 6 A volcano plot of differentially expressed subfamilies of repetitive RNAs labeled. (2C) shows WT mESC and Fto - / - Cumulative distribution and box plots of nuclear LINE1 RNA lifetime in mESCs (inset). (2D) In WT mESCs and Fto - / - Relative enrichment of YTHDC1 to LINE1 RNA in mESCs from CLIP-qPCR. p-values were determined using an unpaired two-sided t-test; error bars and mean ± SD for n = 3 experiments are shown. (2E) shows the relative enrichment of YTHDC1 to LINE1 RNA in WT mESCs and Fto - / - Cumulative distribution of LINE1 RNA transcription rate in mESCs and box plots (inset). (2F) shows the cumulative distribution of LINE1 RNA transcription rate in mESCs after Fto KO. 6 Cumulative distribution plot and box plot (inset) of the difference in transcription rate changes between labeled and unlabeled LINE1 RNA. P values were determined using the Wilcoxon rank sum test. For (C) and (E), p values were determined using the Wilcoxon signed rank test.

[0043] Figure 3 A to Figure 3 D. FTO regulates the expression of LINE1 RNA6 A demethylation affects chromatin status. (3A) Left: DNase I-TUNEL assay showing more closed chromatin after Fto KO. Scale bar, 20 μm. Cell nuclei were counterstained with DAPI. Representative images from 3 independent experiments. Right: WT mESC and Fto - / - Relative TUNEL intensity in mESCs. p-values were determined using an unpaired two-sided t-test. TUNEL intensity was quantified by ImageJ. (3B) WT mESCs (grey) and FTO restored with dCas13b-wtFTO and gRNA targeting LINE1 RNA (red) or control gRNA (blue). - / - TUNEL signal of mESC. (3C) TUNEL signal of mESC - / - Figure 3. H3K4me3 and H3K9me3 profiles at the LINE1 RNA locus, extending from 3.0 kb upstream of the transcription start site (TSS) to 3.0 kb downstream of the transcription end site (TES) in mESCs. (3D) Schematic model showing how Fto KO affects LINE1 RNA abundance and local chromatin state.

[0044] Figure 4 A to Figure 4 D. Fto KO inactivates LINE1-containing genes by suppressing LINE1 RNA within the gene. (4A) Box plots showing the fold change in gene expression after Fto KO from caRNA-seq. Genes were categorized based on the genomic distance between the gene and the nearest LINE1 RNA with at least 10 sequencing reads. (4B) Box plots showing the fold change in expression of LINE1-containing genes after Fto KO and the mRNA expression of the corresponding LINE1 RNA within the gene. 6 A scatter plot of the negative correlation between the expression fold changes of the LINE1 RNA level. The LINE1 RNA within the gene was divided into 100 segments based on its expression fold change after sorting Fto KO. r refers to the Pearson correlation coefficient. The p value was calculated based on the t distribution. (4C) Box plots of gene expression fold change (left) and gene transcription rate fold change (right) from caRNA-seq are shown. The genes were divided into three groups: genes containing downregulated LINE1 RNA, genes close to (<1Mb) downregulated LINE1 RNA, and genes containing other LINE1 RNA. (4D) In WT mESC and Fto - / -Figure 3. H3K4Me3, H3K9Me3, H3K27Ac, and Pol II levels at loci containing downregulated LINE RNAs in mESCs, ranging from 3.0 kb upstream of the TSS to 3.0 kb downstream of the TES. For (A) and (C), p-values were determined using the Wilcoxon rank-sum test.

[0045] Figure 5 A to Figure 5 F. The FTO-LINE1 RNA axis plays a key role in early development. (5A) WT mice and FTO mice at 4 weeks of age - / - Number of GV oocytes in mice. n = 4 (WT) and n = 6 (Fto - / - (5B) WT mice (n=5) and Fto mice at 4 weeks of age. - / - The ratio of SN oocytes to NSN oocytes in WT and Fto mice (n=4). - / - Relative LINE1 RNA expression in oocytes detected by RT-qPCR. (5D) Left: DNase I-TUNEL assay showing more closed chromatin in Fto KO oocytes. Scale bar, 50 μm. Nuclei were counterstained with DAPI. Representative images from three independent experiments. Right: WT oocytes and Fto KO oocytes. - / - Relative TUNEL intensity in oocytes (n=12 per group). pSN: partially enclosed nucleolus; NSN: non-enclosed nucleolus. (5E)Fto P+ / M+ Fto P+ / M- Fto P- / M+ Zygote and Fto P- / M- The implantation rate of zygotes (left) and the embryonic rate at E7.5 (right). P+ / M+ and Fto P- / M- Relative LINE1 RNA expression levels in morulae detected by RT-qPCR. For (A), (C), (D), and (F), p-values were determined using an unpaired, two-sided t-test; error bars and mean ± SD are shown for experiments with n = 3 in (C) and (F). For (B) and (E), p-values were determined using a two-sided z-test.

[0046] Figure 6 A to Figure 6 N. In mESCs, FTO affects the expression of caRNAs 6 A methylation. (6A) WT mice, Fto + / - Mice and Fto - / - Representative images of mice. (6B) shows the expression of WT mESC and Fto- / - Schematic diagram and electrophoresis gel image of PCR products validated in mESC. (6C) Top: Immunoblot analysis of validation of Fto KO in mESC. Bottom: Quantification of WT mESC and Fto by ImageJ. - / - FTO protein levels relative to GAPDH in mESCs. (6D) shows the protein levels of WT mESCs and FTO - / - Immunoblot analysis of cell fractionation efficiency of mESC. (6E to 6G) Quantification of fractionation efficiency of mESC from WT mESC and Ftf by UHPLC-MS / MS - / - mESCs for whole-cell polyadenylated RNA (E), chromatin-associated RNA after rRNA depletion (F), and non-ribosomal RNA from soluble nuclear extracts (neRNA) (G). 6 A / A ratio. m of polyadenylated RNA 6 A levels showed only minimal increase after FtoKO. (6H) Fluorescence imaging of cell nuclei (counterstained with DAPI) and FTO protein (green), indicating that FTO protein is mainly nuclear localized in mESCs. Scale bar, 20 μm. Representative images selected from 3 independent experiments. (6I to 6L) Fluorescence imaging of cell nuclei (counterstained with DAPI) and FTO protein (green), indicating that FTO protein is mainly nuclear localized in mESCs. Scale bar, 20 μm. Representative images selected from 3 independent experiments. - / - Quality analysis of caRNA MeRIP-seq in mESC. (I) 6 The top-ranked consensus motifs identified in peak A. (J) m obtained from n = 3 biological replicates. 6 The overlap of peak A shows high reproducibility. (K) n = m between 3 biological replicates 6 The correlation at level A shows high reproducibility. r refers to the Pearson correlation coefficient. (L)m 6 The distribution of A peaks in different genomic regions including promoter regions, exon regions, intron regions, transcription termination site (TTS) regions and intergenic regions annotated by HOMER. (6M) shows the differentially methylated caRNA mRNAs after Fto KO in mESCs. 6 Volcano plot of peak A. High methylation peaks: 12480; low methylation peaks: 8848. (6N) In WT mESC and Fto - / - Relative total caRNA mRNA from caRNA MeRIP-seq in mESCs 6 A levels of caRNA. For (C), (E) to (G), and (N), p values were determined using an unpaired two-sided t-test; error bars and mean ± SD are shown for n = 3 biological replicates in (C) and (N) and n = 4 biological replicates in (E) to (G).

[0047] Figure 7 A to Figure 7 F. In mESCs, FTO regulates the expression of carRNA 6 A level and expression. (7A) WT mESC and Fto - / - Fto KO in mESCs induces differential expression of carRNA 6 A. The number of peaks. paRNA(+) and paRNA(-) represent sense paRNA and antisense paRNA, respectively. (7B) shows the difference between WT mESC and Fto - / - mRNA expression in mESCs 6 A horizontal box plot. P values were determined using the Wilcoxon signed-rank test. (7C) WT mESC and Fto - / - Differentially expressed mESCs 6 A marks the ratio of carRNA. (7D) shows the fold change of carRNA transcript abundance in mESCs after Fto KO and the mRNA of carRNA. 6 A scatter plot of the negative correlation between the fold changes in the levels of Fto KO based on their sorted m 6 (7E) shows the mRNAs with high methylation expression and down-regulated expression in mESCs after Fto KO. 6 A scatter plot of the negative correlation between the fold change in level and the fold change in expression. These figures indicate the direct effect on carRNA caused by Fto KO. (7F) shows the m of chromatin-associated repetitive RNA (the most sensitive group of carRNA to Fto KO) after Fto removal. 6 A. Schematic model of elevated levels and repressed transcript levels. For (D) and (E), r refers to the Pearson correlation coefficient. P values were calculated based on the t distribution.

[0048] Figure 8 A to Figure 8 I. In mESCs, FTO binds to LINE1 RNA and regulates its m 6 A levels and expression levels. (8A) In WT mESC and Fto - / - In mESCs, the total abundance of LINE1 RNA transcripts on chromatin was quantified using CPM values from caRNA-seq. (8B) In WT mESCs and Fto - / - The relative mRNA expression of LINE1 RNA on chromatin detected by MeRIP-qPCR in mESCs 6A enrichment factor (left), and relative expression of LINE1 RNA on chromatin detected by RT-qPCR (right). (8C) Relative expression of LINE1 RNA and Gapdh (as a control) detected by RT-qPCR in different subcellular fractions. (8D) Fluorescence imaging, intensity profiles, and 2D intensity histograms of LINE 1 RNA (magenta) and FTO protein (green), indicating their colocalization. Scale bar, 10 μm. r refers to the Pearson correlation coefficient determined by Coloc2 in ImageJ. The p value was determined using a one-sample t-test. (8E) CLIP-qPCR of LINE1 RNA bound to FTO in WT mESC (using an antibody against endogenous FTO) and LINE1 RNA bound to IgG (as a negative control) is shown. (8F) Left: FISH showing reduced LINE1 RNA levels after Fto KO. Scale bar, 20 μm. Right: WT mESC and Fto quantified by ImageJ are shown. - / - Box plot of relative FISH intensity in mESC. (8G) In WT mESC and Fto - / - The relative mRNA expression of total LINE1 RNA detected by MeRIP-qPCR in mESCs 6 A enrichment fold (left), and the relative expression of total LINE1 RNA detected by RT-qPCR (right). (8H) Top: shows the expression of WT mESC and Fto - / - Immunoblot analysis of L1ORF1p in mESCs. Bottom: L1ORF1p levels relative to tubulin quantified by ImageJ. (8I) WT mESCs were treated with DMSO (as a control) or FTO inhibitor (FB23-2) for 72 hours. Left: Relative mRNA expression of total LINE1 RNA detected by MeRIP-qPCR. 6 A: Fold enrichment. Right: Relative expression of total LINE1 RNA detected by RT-qPCR. For (D) and (F), cell nuclei were counterstained with DAPI; representative figures were selected from three independent experiments. For (A) to (C) and (E) to (I), p values were determined using an unpaired two-sided t-test; error bars and mean ± SD are shown for n = 3 biological replicates in (A), (C), (E), and (H) and n = 4 biological replicates in (B), (G), and (I).

[0049] Figure 9 A to Figure 9 I. LINE1 RNA mRNA expression in various mammalian tissues 6 A is the physiological substrate of FTO. (9A) m in mouse tissue (left) and human tissue (right)6 Negative correlation between A level and LINE1 RNA expression. (9B) mRNA expression of repetitive RNA in mouse tissues 6 There is a negative correlation between A level and Fto expression. (9C) Among various repetitive RNAs, LINE1 RNA showed high m 6 A level and high expression. (9D) m 6 A Negative correlation between enrichment fold and relative LINE1 RNA expression. (9E) LINE1 RNA m 6 A negative correlation between enrichment fold and relative Fto expression (left), and a positive correlation between relative LINE1 RNA expression and Fto expression in mouse tissues (right). (9F) LINE1 RNA m 6 Negative correlation between A level and FTO expression (left), and positive correlation between LINE1 RNA expression and FTO expression in human tissues (right). (9G) mRNA expression of repetitive RNA in mouse tissues 6 There is no clear correlation between A level and Alkbh5 expression. (9H) LINE1 RNA m 6 There is no clear correlation between the enrichment fold and the relative expression of Alkbh5 (left) or between the relative expression of LINE1 RNA and the relative expression of Alkbh5 (right). (9I) LINE1 RNA m 6 There is no clear correlation between A levels and Alkbh5 expression (left) or between LINE1 RNA expression and Alkbh5 expression (right). 6 A demethylation (13). In HeLa cells, ALKBH5 can remove the 5' UTR m 6 A inhibits LINE1 retrotransposition, whereas FTO has been shown not to affect LINE1 mobility (67). For (A) to (I), correlations are given in scatter plots; r refers to the Pearson correlation coefficient; p values were calculated based on the t distribution. For (A), (B), (C), (F), (G), and (I), RNA-seq datasets and m 6 A MeRIP-seq dataset (13) was used to calculate m 6 A level and expression. For (D), (E) and (H), MeRIP-qPCR and RT-qPCR were used to detect m 6 The enrichment fold and relative expression level of A.

[0050] Figure 10 A to Figure 10 N. In mESCs, Fto KO largely recapitulated the effects of LINE1 ASO treatment. (10A) Shows GSEA of the overall upregulation of LINE1 RNA-targeted genes after Fto KO, as revealed by ChIRP-seq (top) and LINE1 sequence enriched genes (bottom). NES, normalized enrichment score. These findings are similar to those observed after reported LINE1 ASO treatment (27, 28). Together with Figure 1 E, These transcriptome data suggest that FTO acts through similar pathways compared to LINE1 RNA in mESCs. (10B) shows a MA plot of transcriptome changes in mESCs after Fto KO, as analyzed from mRNA-seq. 2C genes are upregulated, and several pluripotency genes are downregulated. (10C and 10D) RNA-seq datasets from GSE100939 (27) were analyzed for LINE1 ASO treatment. (C) A scatter plot showing the fold change in mRNA expression in mESCs under Fto KO or LINE1 ASO conditions. Fto KO and LINE1 ASO showed consistent effects on the transcriptome. Compared to LINE1 ASO treatment, Fto KO resulted in lower 2C gene activation, but higher downregulation of pluripotency genes. (D) A heat map showing consistent changes in most key ESC genes in mESCs under Fto KO conditions and under LINE1 ASO conditions. (10E) Changes in expression of genes that were downregulated (upper panel) or upregulated (lower panel) by Fto KO during the indicated embryonic developmental stages. Genes downregulated after Fto KO were highly expressed in the ESC stage, while genes upregulated were highly expressed in the early 2C stage or 2C stage. The p-value was determined using the Wilcoxon signed-rank test, with ****p<0.0001 indicated for the indicated stage compared to each other stage. The RNA-seq dataset of early embryonic development from GSE66390 (68) was analyzed. (10F) WT mESC and Fto - / - Cell cycle analysis of mESCs. Consistent with LINE1 ASO (27), Fto KO resulted in a decrease in the proportion of cells in S phase. The p-value was determined using a chi-square test. (10G) WT mESCs and Fto KO cells were cultured for three days after initial plating. - / - AP staining of mESCs. Scale bar: 200 µm. Fto KO resulted in only a slight decrease in AP-positive mESCs. (10H) WT mESCs and Fto were cultured for 3 days after passage at the same cell number. - / -Colony morphology of mESC. Scale bar: 200µm. - / - mESCs grew slower than WT mESCs. (10I) Representative images (left) and box plots (right) of increased EB diameter in FtoKO after four days of EB induction in mESCs measured by ImageJ. Scale bar: 200 µm. (10J) Relative expression of total LINE1 RNA quantified by RT-qPCR in WT mESCs treated with reverse complement ASO (RC ASO, as a control) or LINE1 ASO. Error bars and mean ± SD of n = 3 biological replicates are shown. (10K) Fto - / - Proliferation of mESCs (blue solid line) and WT mESCs treated with control ASO (grey solid line) or LINE1 ASO (grey dashed line). - / - Differentiation of mESC (blue solid line) and WT mESC treated with control ASO (grey solid line) or LINE1 ASO (grey dashed line). Proliferation after differentiation induction represents the formation of mouse embryoid bodies (mEBs). (10M) Left: Differentiation of WT mESC and Fto - / - Bright-field and dark-field epifluorescence imaging of mESC-derived chimeras. Scale bar: 1000 µm. Right: Percentage of red fluorescent protein (RFP)-positive cells. n = 3 mice (WT) and n = 6 mice (Fto - / - ) Error bars and mean ± SD. (10N) WT mESC and Fto - / - The proportion of chimeric fetuses generated from mESCs. - / - mESCs showed a poor ability to generate chimeric mice, indicating reduced differentiation potential in vivo. For (I), (J), (M) and (N), p values were determined using an unpaired two-sided t-test. Representative figures were selected from three independent experiments from (G) to (I). Notably, Fto KO resulted in lower 2C gene activation but more downregulation of ESC-highly expressed genes compared to LINE1 ASO treatment; LINE1 ASOs also severely impaired colony formation (27), while Fto KO showed little effect. The inventors also suspect that FTO and ASOs have different targeting specificities; Fto KO and ASO treatment may result in downregulation of different LINE1 RNAs to very different extents.

[0051] Figure 11 A to Figure 11 N. FTO-mediated m 6 A demethylation regulates the abundance of LINE1 RNA subfamily on chromatin. (11A) mRNAs on LINE1 RNAs of different evolutionary ages6 Distribution of peak A. m 6 A shows the enrichment distribution of young LINE1 RNA (<3.5 Myr). (11B) shows the mRNA of old LINE1 and young LINE1 in WT mESC. 6 A level (left) and m 6 A box plot of the fold change in LINE1 levels after Fto KO (right). Young LINE1 showed higher m 6 A level, and a larger m after Fto KO 6 A level changes. (11C) m on LINE1 RNA with different lengths 6 Distribution of peak A. m 6 A shows enrichment distribution in the full-length LINE1 (about 7000 bp). (11D) shows the average m 6 Scatter plot of the negative correlation between A levels and their mean differences in WT mESCs. Each dot represents the mean m within a LINE1 subfamily. 6 A level and mean difference. r refers to the Pearson correlation coefficient. p values were calculated based on t distribution. (11E) In mESCs, the difference m after Fto KO 6 A peak is along the metagenome of LINE1 RNA on chromatin. The high methylation peak is enriched near the 5' end, while the low methylation peak is slightly enriched at the 5' end and in the middle region. (11F) In mESCs, low methylation m 6 Peak A shows the metagenomic profile of the global LINE1 RNA along chromatin. The differential peaks caused by Fto KO showed a distribution similar to the hypomethylated peaks after Mettl3 KO, which supports the idea that Fto mainly demethylates the mRNA deposited by METTL3 in the 5' region of LINE1. 6 A. Model for reversal. caRNA MeRIP-seq data from GSE133600 (22) were analyzed. (11G) Analysis of the indicated young subfamilies of LINE1 RNA on chromatin. Left three columns: Shows the average m 6 Heatmap of m6A levels and expression, and m6A fold change after Fto KO. Right: Shows WT mESC and Fto - / - Heatmap of expression variation between mESCs. The histogram in the heatmap indicates the range of z-scores for each row. Z-scores are calculated as a measure of distance from the mean expressed in standard deviations. Notably, some subfamilies have greater expression differences than mESCs. 6A level difference, indicating a trans-regulatory mechanism. (11H) shows a heat map of the indicated LINE1 subfamilies with consistent RNA expression downregulation when multiple mapped sequencing fragments (top) and single mapped sequencing fragments (bottom) from caRNA-seq were separated, indicating that the bioinformatics analysis of these subfamilies is stable. The z-scores are shown in (G). Figure 2 The young LINE1 subfamily was significantly downregulated after Fto KO in B. In mESCs, the differential m 6 A peak along the metagenomic map of selected FTO-targeted LINE1 subfamily. (11J) Left: In WT mESC and FTO - / - Relative RNA mRNA of FTO-targeted LINE1 subfamily on top-ranked chromatin detected by MeRIP-qPCR in mESCs 6 A. Enrichment fold. Right: In WT mESC and Fto - / - Relative RNA expression of FTO-targeted LINE1 subfamily on selected chromatin regions in mESCs detected by RT-qPCR. - / - mESCs were transfected with dCas13b-FTO (wild-type or inactive mutant) constructs and LINE1 gRNA or control gRNA to regulate the expression of LINE1 RNA. 6 A. Methylation levels. LINE1 gRNA was designed to target the young LINE1 RNA subfamily that is most sensitive to Fto KO. (K) Schematic model of the dCas13b-FTO site-specific demethylation system. (L to N) Left: In WT mESCs and Fto - / - The relative m values of total LINE1 RNA (L), L1Md_Tf (M) and L1Md_A (N) on chromatin detected by MeRIP-qPCR in mESCs 6 A. Enrichment fold. Right: In WT mESC and Fto - / - In mESCs, the relative expression levels of total LINE1 RNA (L), L1Md_Tf (M), and L1Md_A (N) on chromatin were detected by RT-qPCR. - / - Targeting dCas13b-wtFTO to LINE1 RNA in mESCs can restore the expression of both total LINE1 RNA and selected chromatin-targeted LINE1 subfamily FTO. 6A levels and expression amounts. For (J) and (L) to (N), p values were determined using an unpaired two-sided t-test; error bars and mean ± SD are shown for n = 4 biological replicates in (J) and n = 3 biological replicates in (L) to (N).

[0052] Figure 12 A to Figure 12 K. FTO-mediated m 6 A demethylation regulates the lifespan and transcription of the FTO-targeted LINE1 subfamily. (12A) Compared with the control gRNA, Fto - / - The application of dCas13b-wtFTO and gRNA targeting LINE1 RNA (LINE1 gRNA) in mESCs resulted in an increase in the overall LINE1 RNA lifetime. t was calculated using a single-phase decay model in GraphPad Prism. 1 / 2 (12B) Compared with the control gRNA, in Fto - / - The use of dCas13b-wtFTO with LINE1 gRNA in mESCs results in reduced YTHDC1 binding. (12C) Venn diagram showing the overlap between LINE1 RNAs with reduced transcription rates (pink) and LINE1 RNAs with reduced lifespan (blue). Almost half of the LINE1s with reduced lifespan also exhibit reduced transcription rates, suggesting that for a large fraction of LINE1 RNAs, transcription is associated with RNA degradation. (12D) Percentage of young LINE1 subfamilies with reduced transcription rates in LINE1 RNAs. Almost half of the LINE1 RNAs with reduced transcription rates are young LINE1s. (12E) Shows the correlation between WT mESC and FTO from nascent RNA-seq. - / - Cumulative distribution plots and box plots of RNA transcription rates of selected FTO-targeted LINE1 subfamilies in mESCs (inset). P values were determined using the Wilcoxon signed-rank test. LINE1 subfamilies exhibited reduced transcription rates after FTO KO. (12F) shows mESCs after FTO KO from nascent RNA-seq. 6 Cumulative distribution and box plots (inset) of the difference in transcription rates between A-labeled and unlabeled ERVK (left) or Alu (right). 6 Minimal differences were observed between A-labeled and unlabeled transcripts, suggesting that these repeat RNAs may be indirectly affected by Fto or LINE1 RNA through transregulation. P values were determined using the Wilcoxon rank sum test. (12G) shows the p-values of WT mESC and Fto - / -ChIRP-qPCR of relative DNA binding of LINE1 RNA in mESCs. Fto KO resulted in reduced DNA binding of total LINE1 RNA and selected Fto-targeted LINE1 subfamilies. Malat1 was used as a negative control. (12H) shows the relative DNA binding of WT mESCs and Fto - / - DRIP-qPCR of relative levels of LINE1 RNA:DNA hybrids in mESCs. Fto KO resulted in reduced R-loop formation for both total LINE1 RNA and selected Fto-targeted LINE1 subfamilies. The reduction in DNA association and R-loop formation was consistent with a decrease in LINE1 RNA transcription rate. (12I to 12K) For total LINE1 RNA and selected Fto-targeted LINE1 subfamilies, Fto KO resulted in reduced R-loop formation for both total LINE1 RNA and selected Fto-targeted LINE1 subfamilies compared to control gRNA. - / - Application of dCas13b-wtFTO and LINE1 gRNA in mESCs resulted in increased transcription rate (I), increased DNA binding (J), and restored R-loop formation (K). For (B), (G), (H), (J), and (K), p values were determined using an unpaired two-sided t-test; error bars and mean ± SD of n = 3 biological replicates are shown.

[0053] Figure 13 A to Figure 13 J. In mESCs, FTO regulates global transcription rate and chromatin accessibility. (13A) Left: 5-ethynyluridine (EU) labeling followed by fluorescence imaging demonstrates a reduction in overall nascent RNA synthesis after Fto KO. Scale bar, 20 μm. Nuclei were counterstained with DAPI. Right: Quantification of WT mESCs and Fto by ImageJ. - / - Box plot of relative EU intensity in mESC. (13B) In WT mESC and Fto - / -Relative 4-thiouridine (4sU) / A ratio in total RNA quantified by UHPLC-MS / MS in mESC. (13C) Left: EU labeling followed by fluorescence imaging demonstrated that overall nascent RNA synthesis decreased after FTO inhibitor treatment. Right: Box plots of relative EU intensity are shown. (13D) Left: DNase I-TUNEL assay followed by fluorescence imaging demonstrated that chromatin was more closed after FTO inhibitor treatment. Right: Box plots of relative TUNEL intensity are shown. For (C) and (D), WT mESCs were treated with FTO inhibitor (FB23-2) or DMSO (as a control) for 72 hours. Scale bar, 20 μm. Cell nuclei were counterstained with DAPI. EU intensity and TUNEL intensity were quantified by ImageJ. (13E) DNase I-TUNEL assay followed by flow cytometry shows that in WT mESCs, chromatin is more closed after LINE1 RNA knockdown (purple) with LINE1 ASO compared to RC ASO (gray). LINE1 ASO causes less chromatin closure compared to Fto KO. (13F) Fto cells treated with dCas13b-mutFTO and LINE1 gRNA (red) or control gRNA (blue) - / - TUNEL signals of mESCs were compared with those of WT mESCs (grey). No significant changes in TUNEL signals were observed. (13G to 13I) WT mESCs were treated with FTO inhibitor (FB23-2) for 48 hours. (G) Relative mRNA expression of LINE1 RNA detected by MeRIP-qPCR. 6 A enrichment fold. (H) Relative expression of LINE1 RNA detected by RT-qPCR. (I) Left: DNase I-TUNEL assay followed by fluorescence imaging showed gradual chromatin closure. Scale bar, 20 μm. Right: Box plot showing relative TUNEL intensity quantified by ImageJ. (13J) A schematic model of the plausible mechanism by which FTO inhibition leads to LINE RNA repression and chromatin closure is shown. For (A), (C), (D), and (I), representative figures were selected from three independent experiments. For (A), (C), (D), and (G) to (I), p values were determined using an unpaired two-sided t-test; for (G) and (H), error bars and mean ± SD of n = 3 biological replicates are shown.

[0054] Figure 14 A to Figure 14 E. WT mESC and Fto - / - ATAC-seq and overall changes in histone marks in mESCs. (14A) shows the quantification of WT mESCs and Fto by RPKM.- / - Density plot of total ATAC-seq signal at the center of the peak in mESC. (14B) shows WT mESC and Fto - / - Circos plot of ATAC peaks along the genome in mESC. (14C) Volcano plot of differential ATAC peaks after Fto KO in mESC is shown. Significantly increased peaks are shown in red (p<0.01: 889; FDR<0.05: 164), and significantly decreased peaks are shown in blue (p<0.01: 1397; FDR<0.05: 251). The original peaks were merged into a joint peak set, resulting in more significantly decreased peaks. The p-values of the differential peaks were determined by FeatureCounts and DESeq2. (14D) Gene ontology (GO) analysis of the top-ranked enriched biological processes (blue) and enriched molecular functions (red) of the acquired closed regions. The acquired closed regions are composed of genes from WT mESC and Fto. - / - 1 kb sliding window (57) in ATAC-seq of mESCs was used to define the region. After Fto KO, 4683 acquired occlusion regions were identified. (14E) Left: WT mESCs and Fto - / - Immunoblot analysis of histone modification levels in mESCs. Right: Quantification of histone H3 levels in WT mESCs and Fto by ImageJ. - / - Histone modification levels in mESCs. Consistent with the finding of more closed chromatin, Fto KO resulted in a slight decrease in active markers and an increase in repressive markers. P values were determined using an unpaired two-sided t-test; error bars and mean ± SD of n = 3 independent immunoblots are shown.

[0055] Figure 15 A to Figure 15C. Genomic signatures of acquired occlusion regions following Fto KO derived from ATAC-seq. (15A to 15C) Cross-analysis of histone marks and transcription factors from ATAC-seq and ChIP-seq datasets. ChIP-seq binding sites sorted by acquired occlusion regions from ATAC-seq are plotted in a heatmap view. (15A) Acquired occlusion regions following Fto KO from ATAC-seq are enriched for H3K4Me1, H3K4Me3, H3K27Ac, Pol II, YY1, and P300, but not for H3K9Me3, H3K27Me3, or H3K36Me3 regions identified from ChIP-seq in WT mESCs. YY1: Yin-Yang 1, transcription factor; EP300: E1A-binding protein P300, transcriptional coactivator. Previous studies have shown that both proteins can be recruited by caRNAs to promote transcription (22, 29, 30). METTL3 binding sites are also enriched in acquired occlusion regions after Fto removal, which may explain some of the hypomethylated m 6 Peak A. (15B) FtoKO-induced acquired closure regions were also enriched for H3K4Me3, H3K27Ac, and reduced Pol II binding, YY1 binding, and EP300 binding, which were found to be reduced after Fto KO from ChIP-seq. Consistently, regions with reduced METTL3 chromatin binding after Fto removal were enriched for FtoKO-induced acquired closure regions. Taken together, these findings are consistent with the observed role of FTO in mESC transcriptional activation. (15C) FtoKO-induced acquired closure regions were also enriched for H3K4Me3, H3K27Ac, and increased YY1 binding and EP300 binding, which were found to be increased after Mettl3 KO, suggesting that these effects are m 6 A-dependent. For (A) and (C), datasets from ENCODE or GSE133600 (22) were used.

[0056] Figure 16 A to Figure 16 H. FTO-mediated mRNA expression of LINE1 RNA 6 A. Demethylation regulates local chromatin states. (16A to 16C) show the ChIP signal profile at the LINE1 RNA locus from 3.0 kb upstream of the transcription start site (TSS) to 3.0 kb downstream of the transcription end site (TES). (A) WT mESC and Fto - / - mESCs contain m 6A. Schematic diagram of H3K27Ac levels, H3K4Me1 levels, H4K20Me3 levels, and RNA polymerase II (Pol II) levels at the locus of the labeled LINE1 RNA. After Fto KO, H3K27Ac levels and Pol II levels were reduced. (B to C) WT mESC and Fto - / - Figure 16D shows a box plot of the fold change of H3K4Me3 levels, H3K27Ac levels, and H3K9Me3 levels in the TSS regions of the LINE1 subfamily in mESCs after Fto KO. After Fto KO, the loci of the selected FTO-targeted LINE1 subfamily showed an increase in H3K4Me3 levels and H2K27Ac levels, while the H3K9Me3 level decreased. Using mESC without Fto KO, the H3K4Me3 levels and H2K27Ac levels of the selected FTO-targeted LINE1 subfamily increased, while the H3K9Me3 level decreased. 6 The LINE1 subfamily L1M2b locus of A was used as a negative control. After Fto KO, the L1M2b locus and the locus of the ERVK subfamily IAPez-int (23-25) that was shown to be regulated by METTL3 showed negligible or opposite changes in H3K4Me3 levels, H2K27Ac levels, and H3K9Me3 levels. (16E) Compared with the control gRNA, Fto - / - Application of dCas13b-wtFTO with LINE1 gRNA in mESCs resulted in increased H3K4Me3 and H3K27Ac levels and decreased H3K9Me3 levels at the total LINE1 RNA locus and at loci of selected FTO-targeted LINE1 subfamily. (16F) In WT mESCs and FTO - / - In mESCs, the chromatin binding maps of YY1 (top) and EP300 (bottom) at the center of their peaks and the 0.5 kb regions on both sides. Fto KO leads to an overall decrease in YY1 binding and EP300 binding. (16G) shows the chromatin binding of YY1 and EP300 in mESCs. 6 A: Box plot showing that chromatin binding at the LINE1 RNA locus is reduced after Fto KO, indicating that FTO-mediated mRNA expression of LINE1 RNA is reduced. 6 A demethylation regulates local chromatin by affecting transcription factor binding. This supports the following model: in mESCs, FTO-mediated LINE1 RNAm6 The transcriptional activation of A demethylation involves the binding of EP300 to YY1, thereby promoting the opening of local chromatin (22, 29, 30). (16H) shows the expression of WT mESC and Fto - / - CLIP-qPCR of LINE1 RNA binding to YY1 (left) and EP300 (right) in mESCs. IgG was used as a normalization control. Fto KO resulted in reduced YY1 and EP300 binding to total LINE RNA and selected FTO-targeted LINE1 subfamilies. Consistent with previous reports (22, 29, 30), reduced transcription factor binding to LINE1 RNA may be responsible for its reduced local chromatin binding. For (D) and (G), p values were determined using the Wilcoxon signed-rank test. For (E) and (H), p values were determined using an unpaired two-sided t-test; error bars and mean ± SD of n = 3 biological replicates are shown.

[0057] Figure 17 A to Figure 17 G. LINE1 RNA is a functionally relevant substrate of FTO in EB differentiation and self-renewal of mESC. (17A) In WT mESC and FTO - / - Relative LINE1 RNA expression (left) and relative Fto expression (right) measured by RT-qPCR on day 0 and day 6 of EB differentiation of mESC. After EB differentiation, both LINE1 RNA and Fto increased in expression; in mESCs after 6 days of EB differentiation, LINE1 RNA showed a greater decrease after Fto KO. The p value was determined using an unpaired two-sided t-test; error bars and mean ± SD of n = 4 biological replicates are shown. (17B) shows the relative LINE1 RNA expression in WT mESC and Fto on day 0 and day 6 during EB differentiation. - / - Heatmap of the expression of key differentiation markers in mESCs measured by RT-qPCR. After 6 days of EB differentiation, it was observed that the expression of pluripotency markers Oct4 and Sox2 decreased after Fto KO, while most markers showed changes in higher expression levels. (17C and 17D) WT mESCs (solid gray line), and Fto restored by dCas13b-wtFTO (C) or dCas13b-mutFTO (D) with LINE1 gRNA (dashed blue line) or control gRNA (solid blue line) - / - mESC differentiation. - / - Targeting dCas13b-wtFTO to LINE1 RNA in mESCs can partially restore the expression of Fto - / -Induction of differentiation observed in mESCs. Proliferation after differentiation induction represents the formation of mEBs. (17E) LINE1 ASO treatment of WT mESCs or Fto - / - Hierarchical clustering of the expression changes of differentiation markers in the dCas13b restoration experiment of mESCs. WT mESCs treated with RC ASO and FTO restored with dCas13b-wtFTO and LINE1 gRNA. - / - mESCs were clustered together using Euclidean distance and average linkage methods. (17F and 17G) WT mESCs (grey solid line), and FTOs restored by dCas13b-wtFTO (F) or dCas13b-mutFTO (G) with LINE1 gRNA (blue dashed line) or control gRNA (blue solid line). - / - mESC proliferation. - / - Targeting dCas13b-wtFTO to LINE1 RNA in mESCs could partially restore the induced-reduced proliferation.

[0058] Figure 18 A to Figure 18 H. FTO regulates the transcription and local chromatin state of LINE1-containing genes. (18A) Genes are divided into two groups: genes containing the indicated LINE1 subfamily and genes close to (<1Mb) the indicated LINE1 subfamily. Box plots of gene expression fold changes in mESCs after Fto KO from caRNA-seq are shown. After Fto KO, selected LINE1-containing genes of the Fto-targeted LINE1 subfamily showed decreased expression compared to genes without these LINE1 subfamilies. (18B) Scatter plots showing the positive correlation between the expression fold changes of LINE1-containing genes in mESCs from caRNA-seq and the corresponding intragenic LINE1 RNA after Fto KO. Intragenic LINE1 RNAs were divided into 100 segments based on their sorted transcript abundance fold changes after Fto KO. r refers to the Pearson correlation coefficient. p-values were calculated based on the t-distribution. (18C) WT mESCs and Fto from nascent RNA-seq are shown. - / -Cumulative distribution plot and box plot of transcription rates of LINE1-containing genes in mESC (inset). The transcription rates of LINE1-containing genes were significantly reduced after Fto KO. (18D) Box plot showing the fold change in gene transcription rates in mESCs after Fto KO from nascent RNA-seq. Genes were grouped based on the distance between the gene and the nearest LINE1 RNA expressed with at least 10 sequenced fragments measured. "0kb" represents LINE1 RNA located within the main body of the gene along the genome (genes are referred to as LINE1-containing genes). Fto KO resulted in a decrease in the transcription rate of LINE1-containing genes compared to genes in other groups. (18E) Genes were classified as (A). Box plots show the differences in gene transcription rates in mESCs after Fto KO from nascent RNA-seq. After Fto KO, selected LINE1-containing genes of the FTO-targeted LINE1 subfamily exhibited significantly reduced transcription rates compared to genes without these LINE1 subfamilies. (18F) shows a box plot of the fold change in the levels of H3K4Me3, H3K27Ac, and H3K9Me3 in the TSS regions of intragenic LINE1 and intergenic LINE1 in mESCs from ChIP-seq after Fto KO. After Fto KO, the intragenic LINE1 RNA locus showed a more significant decrease in H3K27Ac levels, a more significant increase in H3K9Me3 levels, and a more significant decrease in H3K4Me3 levels in the intergenic LINE1 RNA locus. The p value was determined using the Wilcoxon signed rank test. (18G) shows the fold change in the lifespan of mESCs after Fto KO from nuclear RNA lifespan sequencing (left) and from nascent RNA-seq. 6 A Cumulative distribution plot and box plot (inset) of the difference in transcription rates between marked intragenic LINE1 RNA and intergenic LINE1 RNA changes (right). Intragenic LINE1 RNAs showed a larger reduction in lifespan, while intragenic and intergenic LINE1 RNAs showed comparable reductions in transcription rates after Fto KO. (18H) Shown is a map of ChIP signals for the indicated histone marks from 3.0 kb upstream of the TSS to 3.0 kb downstream of the TES at the loci containing genes of the FTO-targeted LINE1 RNA subfamily. Fto KO resulted in decreased H3K4Me3 levels and H3K27Ac levels, and increased H3K9Me3 levels at the loci containing selected genes of the FTO-targeted LINE1 RNA subfamily.

[0059] Figure 19 A to Figure 19G. IGV peak plots of methylation, abundance, and local chromatin state of selected downregulated LINE1 loci and genes containing downregulated LINE1 are shown. (19A) A Venn diagram showing the overlap between genes containing downregulated LINE1 RNA and downregulated genes in mESCs after Fto KO from caRNA-seq. (19B) GO analysis of downregulated genes containing downregulated LINE1 RNA (1434 genes were identified). GO terms related to transcription, cell differentiation, and neuronal / cardiac development were enriched, indicating that these downregulated genes containing downregulated LINE1 RNA are responsible for the ESC phenotype and developmental phenotype caused by Fto KO. (19C to 19F) ChIP-seq, caRNA-seq, and caRNA mRNAs of (C) Esrrb, (D) Nek5, (E) Phf3, and (F) Zfp982, which are selected downregulated genes containing downregulated LINE1 RNA, are shown. 6 IGV tracks of A-seq. RNA profiles and m 6 A spectrum is divided into forward strand (f) and reverse strand (r). For the LINE1RNA in the gene, m 6 A levels increased and expression on chromatin decreased. Decreased H3K4me3 levels, H3K27ac levels, and Pol II levels were observed at the LINE1 locus and TSS regions within genes containing LINE1 genes. Except for Phf3, all genes also showed increased H3K9Me3 levels at the LINE1 locus. Esrrb is known to play an important role in the pluripotency network of mESCs (69). All of the other three genes have important functions in mESCs or early development. (19G) Decreased transcription rates from nascent RNA-seq were observed for all four genes.

[0060] Figure 20 A to Figure 20 J. Application of dCas13b-wtFTO reversed the lifespan changes of LINE1 RNA in selected genes and the transcriptional changes of LINE1-containing genes. (20A) shows WT mESC and FTO - / - Fto in mESC (left) and restored by dCas13b-wtFTO and LINE1 gRNA or control gRNA - / -CLIP-qPCR of YTHDC1 binding to L1MD3 (in Essrb) in mESCs (right). IgG was used as a normalization control. Fto KO resulted in increased YTHDC1 binding to L1MD3 (in Essrb), which was reversed by targeting dCas13b-wtFTO to LINE1 RNA. (20B) Fto KO in WT mESCs (grey) and restored with dCas13b-wtFTO and LINE1 gRNA (red) or control gRNA (blue). - / - Lifespan of L1MD3 (in Essrb) in mESCs. Fto KO resulted in a reduction in the lifespan of L1MD3 (in Essrb), which was reversed by targeting dCas13b-wtFTO to LINE1 RNA. (20C) Relative changes in transcription rate, H3K4Me3 levels, H3K9Me3 levels, H3K27Ac levels, and Pol II levels of Esrrb after application of dCas13b-wtFTO with LINE1 gRNA or control gRNA. Targeting dCas13b-wtFTO to LINE1 RNA resulted in increased gene transcription rate, increased local H3K4me3 levels, H3K27ac levels, and pol II binding levels, and decreased local H3K9Me3 levels. (20D) shows the relative changes in transcription rate, H3K4me3 levels, H3K27ac levels, and pol II binding levels of WT mESCs and Fto - / - Fto in mESC (left) and restored by dCas13b-wtFTO and LINE1 gRNA or control gRNA - / -CLIP-qPCR of YTHDC1 binding to L1MdTf_I (in Phf3) in mESCs (right). IgG was used as a normalization control. Fto KO resulted in increased YTHDC1 binding to L1MdTf_I, which could be reversed by targeting dCas13b-wtFTO to LINE1 RNA. (20E) Lifespan of L1MdTf_I (in Phf3) in Fto- / - mESCs and WT mESCs (gray) restored by dCas13b-wtFTO and LINE1 gRNA (red) or control gRNA (blue). Fto KO resulted in a decrease in the lifespan of L1MdTf_I (in Phf3), which was reversed by targeting dCas13b-wtFTO to LINE1 RNA. (20F) Only minor changes in the lifespan of Esrrb transcripts or Phf3 transcripts were observed from nuclear RNA lifespan sequencing. (20G) From nuclear RNA lifespan sequencing, we observed a reduction in the lifespan of L1MdA_III (in Nek5) and L1MdF_I (in Zfp982). The present inventors were able to design primers for these two subfamilies of LINE1 RNA, but due to multiple targeting, we were unable to design primers specifically targeting the L1MdA_III locus contained in Nek5 or the L1MdF_I locus contained in Zfp982. (20H) Compared with the control gRNA, in Fto - / - Application of dCas13b-wtFTO with LINE1 gRNA in mESCs resulted in increased transcription rates of Nek5, Phf3, and Zfp982. (20I) Compared with control gRNA, in Fto - / - Application of dCas13b-wtFTO with LINE1 gRNA in mESCs resulted in increased H3K4Me3 and H3K27Ac levels at the Nek5, Phf3, and Zfp982 loci, while decreased H3K9Me3 levels. (20J) - / - Relative expression levels of Dub1, Zscan4, and MERVL RNA after application of dCas13b-wtFTO with LINE1 gRNA or control gRNA in mESCs. All three targets have been reported to be repressed by scaffold-like transregulation of LINE1 RNA (27). - / - Targeting dCas13b-wtFTO to LINE1 RNA in mESCs reduced their expression. Note that the 2C genes Dub1 and Zscan4 do not contain LINE1 RNA. MERVL is a 2C-specific transposon that is also repressed by LINE1 RNA. This result supports the FTO-mediated mESC 6A. Scaffolding transregulation of LINE1 RNA by demethylation. For (A), (C), (D), (I), and (J), p values were determined using an unpaired, two-sided t-test; error bars and mean ± SD of n = 3 biological replicates are shown. For (B) and (E), t values were calculated using a one-phase decay model in GraphPad Prism. 1 / 2 .

[0061] Figure 21 A to Figure 21 L. FTO regulates the expression of LINE1 in brain tissue 6 A level and RNA level, as well as chromatin status. (21A) Left: from WT mice and Fto - / - Relative LINE1 RNA mRNA in total RNA isolated from mouse cerebellum 6 A: Enrichment fold. Right: Relative LINE1 RNA expression in the same sample. (21B and 21C) Left: In samples from WT mice and Fto - / - DNase I-TUNEL assay was performed in mouse cerebellum sections, followed by fluorescence imaging at 10× (B) or 40× (C) fields of view, indicating more closed chromatin. Scale bars, 120 μm for (B) and 30 μm for (C). Right: Bar graph (B) or box plot (C) showing relative TUNEL intensity. Relative TUNEL intensity was quantified by ImageJ as the average of all cells in one image (B) or the signal in each cell nucleus (C). (21D) Left: WT mice and Fto - / - Relative LINE1 RNA mRNA in total RNA isolated from mouse hippocampus 6 A. Enrichment fold. Right: Relative expression of LINE1 RNA in the same samples detected by RT-qPCR. (21E and 21F) - / - DNase I-TUNEL assay was performed in mouse hippocampal sections, followed by fluorescence imaging at 10× (E) or 40× (F) fields of view, indicating more closed chromatin. Scale bar, 120 μm for (E) and 30 μm for (F). Right: Bar graph (E) or box plot (F) showing relative TUNEL intensity. Relative TUNEL intensity was quantified by ImageJ as the average of all cells in one image (E) or the signal in each cell nucleus (F). (21G) Left: WT aNSC and Fto - / - Relative LINE1 RNA mRNA in total RNA isolated from aNSCs 6A enrichment factor. Right: Relative LINE1 RNA expression in the same sample. (21H) Left: DNase I-TUNEL assay followed by fluorescence imaging showed that the chromatin in aNSC measured after Fto KO was more closed. Right: Box plot showing relative TUNEL intensity. (21I) Left: EU labeling followed by fluorescence imaging showed that the overall nascent RNA synthesis in aNSC was reduced after FTO KO. Right: Box plot showing relative EU intensity. For (H) and (I), the detection values are from WT aNSC and Fto - / - aNSC. Scale bar, 20 μm. The relative intensity of EU or TUNEL relative to DAPI was quantified in each cell nucleus by ImageJ. (21J) Top: Figure 2 shows the relative intensity of EU or TUNEL from WT mice and Fto - / - Immunoblot analysis of H3K4Me3 levels in the cerebellum and hippocampus of mice; Lower panel: H3K4Me3 protein levels relative to histone H3 quantified by ImageJ. (21K) WT aNSCs and Fto cells before (proliferation) and after (differentiation) differentiation. - / - Relative LINE1 RNA expression in total RNA of aNSC. Consistent with the observations in EB differentiation of mESC, LINE1 RNA showed increased expression after differentiation and was more significantly reduced in differentiated aNSCs after Fto KO. (21L) Top: Immunoblot analysis of FTO levels in WT aNSCs before (proliferation) and after (differentiation) differentiation is shown. Bottom: FTO protein levels relative to GAPDH quantified by ImageJ. p-values were determined using an unpaired two-sided t-test; error bars and mean ± SD are shown for n = 4 biological replicates in (A), (D), (G) and (K), and for n = 3 biological replicates in (B), (E), (J) and (L). For (A), (D) and (J), mESCs were detected by MeRIP-qPCR and RT-qPCR, respectively. 6 Fold enrichment and relative expression levels for A. For (B), (C), (E), (F), (H), and (I), cell nuclei were counterstained with DAPI; representative images were selected from three independent experiments.

[0062] Figure 22 A to Figure 22 K. The FTO-LINE1 RNA axis plays an important role in oocyte development. (22A) Percentage of embryonic genotypes from heterozygous crosses. - / - The embryo ratio is significantly lower than the theoretical value. The p value was determined using the chi-square test for 221 embryos collected from n=5 independent experiments. (22B) from WT males and WT females and Fto - / -The number of pups per cross (left) and the number of offspring surviving 24 hours later (right) in female crosses. After mating with WT male mice, Fto KO mothers showed more mortality and reduced number of pups, and all surviving Fto + / - The pups died shortly after birth. Error bars and mean ± SEM of n = 6 pairs of mice are shown. (22C) Pups from 4-week-old WT mice and Fto - / - Representative images of mouse ovaries (left); from WT mice at 4 weeks of age (middle) or 7 weeks of age (right) and Fto - / - HE staining of mouse ovary. - / - The mouse ovaries showed defects of slightly smaller size and reduced number of primordial follicles. (22D) The expression of Fto (broken line) and LINE1 RNA (bar) quantified by CPM values throughout oocyte development from the total RNA-seq dataset (GSE75738) (70) were analyzed. (22E) The expression of Fto (broken line) and LINE1 RNA (bar) were quantified by CPM values throughout oocyte development. - / - Box plots of LINE1 RNA levels (RPKM) from RNA-seq of mouse GV oocytes and MII oocytes that were significantly reduced compared to WT controls. P values were determined using the Wilcoxon signed-rank test. (22F) Scatter plots showing the fold changes in LINE1 subfamily expression in GV oocytes or MII oocytes after Fto KO. LINE1 subfamilies that were significantly downregulated in mESCs after Fto KO are marked in blue. Compared to WT controls, LINE1 subfamilies from Fto - / - The changes in RNA expression of LINE1 subfamily in mouse oocytes showed a pattern consistent with the changes in RNA expression caused by Fto KO in mESCs. (22G) From 4-week-old Fto - / - The number of MII oocytes collected from mice and WT controls. - / - MII numbers in mice show great variability. n = 7 Fto are shown - / - Error bars and mean ± SEM of mice and n=6 WT controls. (22H) Left: shows the - / - Fluorescence imaging of meiotic defects in mouse MII oocytes compared to WT controls. Scale bar, 50 μm. Right: Compared to WT controls, from Fto - / - The percentage of chromosome misalignment and spindle disassembly in mouse MII oocytes is increased. Error bars and mean ± SD of three independent experiments are shown. (22I) shows Fto - / -Scatter plots showing the positive correlation between LINE1-containing genes and the fold change in transcript abundance of the corresponding intragenic LINE1 RNA relative to WT controls in GV oocytes (left) and MII oocytes (right) of mice. Intragenic LINE1 RNAs were divided into 100 bins based on their ranked transcript abundance fold change after Fto KO. r refers to the Pearson correlation coefficient. p-values were calculated based on the t-distribution. (22J) shows the Fto from RNA-seq. - / - Box plots of fold change in gene expression relative to WT controls in GV oocytes (left) and MII oocytes (right) of mice. Genes were divided into three groups: genes containing downregulated LINE1 RNA (denoted as genes containing downregulated LINE1, “downregulated” and “contains”), genes close to (<1 Mb) downregulated LINE1 RNA (“downregulated”), and genes containing non-downregulated LINE1 RNA (“contains”). Fto KO resulted in a significant decrease in the expression of genes containing downregulated LINE1 RNA compared to other genes. The p-value was determined using the Wilcoxon rank sum test. (22K) - / - GO analysis of downregulated genes containing downregulated LINE1 RNA in mouse GV oocytes or MII oocytes compared to WT controls. For (B), (G), and (H), p values were determined using an unpaired two-sided t-test.

[0063] Figure 23 A to Figure 23 R. FTO-mediated LINE1 RNA mRNA expression during embryonic development 6 A demethylation plays an important role. (23A) shows a schematic model of the mating workflow. (23B) from WT MII oocytes and Fto - / - The in vitro fertilization (IVF) rate (expressed as a percentage) of hybridization between spermatozoa. - / - Sperm activity decreased, but most fertilized zygotes can still develop to the blastocyst stage. Since the loss of Fto in sperm will lead to a decrease in in vitro fertilization rate, the inventors used intracytoplasmic sperm injection (ICSI) technology to fertilize oocytes. (23C) Fto P+ / M+ Fto P+ / M- Fto P- / M+ Zygote and Fto P- / M-Percentage of zygotes progressing to blastocysts (E3.5, left; E4.5, right). Paternal, maternal, or double knockout of Fto all resulted in a mild block in blastocyst progression. (23D) Representative images of implantation defects following Fto loss are shown. Paternal or maternal loss of Fto resulted in reduced implantation rates. Scale bars: 1000 µm for decidua, 500 µm for embryos. Representative images were selected from two independent experiments. No E7.5 embryos were obtained from embryos with double knockout of both Fto alleles. (23E) Fto from RNA-seq is shown. P+ / M+ LINE1 RNA levels (RPKM) in morulae and Fto P- / M- (23F) Scatter plot showing the fold change in RNA expression of LINE1 subfamily in mESC or morula after Fto KO. LINE1 subfamily in mESC that was significantly downregulated after Fto KO is marked in blue. P- / M- Morula and Fto P+ / M+ Changes in RNA expression of LINE1 subfamily between morulae and Fto - / - The changes in RNA expression of LINE1 subfamily between mESC and WT mESC are consistent. (23G) shows the expression of RNA from Fto P+ / M+ Morula and Fto P- / M- RT-qPCR of elevated Zscan4 RNA levels and MERVL RNA levels in morulae. Error bars and mean ± SD of n = 3 experiments are shown. (23H) Fto P- / M- The MERVL RNA level (RPKM) in morulae was significantly correlated with the Fto P+ / M+ Box plot of elevated levels of Ft in morulae compared to morulae. P values were determined using the Wilcoxon signed rank test. (23I) shows the Ft from RNA-seq. P- / M- Gene expression in morula and Fto P+ / M+ Figure 23J shows the box plot of the fold change of LINE1 RNA compared to morulae. The genes were divided into three groups: genes containing downregulated LINE1 RNA (indicated as genes containing downregulated LINE1, "downregulated" and "containing"), genes close to (<1 Mb) downregulated LINE1 RNA ("downregulated"), and genes containing non-downregulated LINE1 RNA ("containing"). Fto KO resulted in a significant decrease in the expression of genes containing downregulated LINE1 RNA compared to other genes. The p value was determined using the Wilcoxon rank sum test. (23J) shows the expression of Fto in genes containing downregulated LINE1 RNA. P- / M-Scatter plot showing the positive correlation between LINE1-containing genes in morulae and the corresponding intragenic LINE1 RNA expression fold change compared to Fto P+ / M+ morulae. Intragenic LINE1 RNA was divided into 100 segments based on their transcript abundance fold change after sorting Fto KO. r refers to the Pearson correlation coefficient. p-values were calculated based on the t-distribution. (23K) Fto from RNA-seq P+ / M+ Morula and Fto P- / M- Expression of selected down-regulated genes containing down-regulated LINE1 RNA in morulae. Lin28b, Tet2, and Gsk3b were identified as genes containing down-regulated LINE1 RNA in both mESCs and morulae, supporting the model that Fto ablation leads to delayed 2C exit and developmental defects. P values were determined using DESeq2. (23L) P- / M- Morula and Fto P+ / M+ GO analysis of genes containing down-regulated LINE1 RNA compared to those down-regulated in morulae. (23M) Schematic model of the dcas13b-wtFTO restoration experiment during early development (created using BioRender): dcas13b-wtFTO was microinjected into Fto along with control gRNA or LINE1 gRNA. - / - MII oocytes. - / - After ICSI and in vitro development of sperm, Fto P- / M- Morulae (labeled with LINE1 gRNA, red, or control gRNA, blue; based on the microinjection procedure at the MII stage) were used for subsequent experiments. (23N) shows Fto treated with dCas13b-wtFTO and LINE1 gRNA or control gRNA at different stages. P+ / M+ Embryo, Fto P- / M- Embryos and FTO P- / M- Representative images of embryos. Representative images were selected from two independent experiments. (23O to 23R) Fto P- / M- Morulae were derived from Fto cells injected with dCas13b-wtFTO and control gRNA (blue) or LINE1 gRNA (red). - / - MII oocytes. (O) Relative expression of LINE1 in total RNA isolated from morulae detected by RT-qPCR. - / - Targeting dCas13b-wtFTO to LINE1 RNA in MII oocytes can enhance FTO expression after development to the morula stage. P- / M-LINE1 and MERVL RNA expression in embryos. (P) Left: DNase I-TUNEL assay followed by fluorescence imaging showed that Fto treated with LINE1 gRNA P- / M- Chromatin in morulae is more open compared to that treated with control gRNA. Scale bar, 20 μm. Nuclei were counterstained with DAPI. Representative images were selected from n=8 morulae (control gRNA) and n=10 morulae (LINE1 gRNA). Right: Box plot showing relative TUNEL intensity in morulae. Relative TUNEL intensity was quantified by ImageJ. - / - MII targeting LINE1 RNA with dCas13b-wtFTO resulted in a chromatin-dependent P- / M- Morulae are more open, which is associated with increased LINE1 RNA expression. (Q) shows the expression of Fto treated with LINE1 gRNA. P- / M- RT-qPCR analysis of the reduced RNA levels of Zscan4 and MERVL in total RNA isolated from morulae compared with those treated with control gRNA. - / - Targeting LINE1 RNA with dCas13b-wtFTO in MII oocytes reduced the RNA expression of Zscan4 and MERVL after development to the morula stage. (R) Relative expression of selected down-regulated genes containing down-regulated LINE1 RNA in total RNA isolated from morulas by RT-qPCR. n = average of 2 biological replicates. These three targets are Fto P- / M- Morula and Fto P+ / M+ Morula compared with Fto - / - Down-regulated genes containing down-regulated LINE1 RNA shared by mESCs compared with WT mESCs. - / - Targeting dCas13b-wtFTO to LINE1 RNA in MII oocytes increased Fto P- / M- Expression levels of Lin28b, Tet2, and Gsk3b in morulae. For (E) and (H), p values were determined using the Wilcoxon signed-rank test. For (G) and (O) to (R), p values were determined using an unpaired two-sided t-test; the average values of n = 2 experiments in (O), (Q), and (R) are shown.

[0064] Figure 24 A to Figure 24 C. Fto KO leads to accelerated carRNA degradation and transcriptional inhibition. (24A) shows WT mESC and Fto - / -Cumulative distribution of carRNA lifespan changes in mESC and box plots (inset). CarRNAs in each group showed reduced lifespan after FtoKO. (24B) shows the difference between WT mESC and Fto - / - Cumulative distribution of carRNA transcription rates in mESC and box plots (inset). CarRNAs in each group showed reduced transcription rates after Fto KO. (24C) shows m 6 Cumulative distribution and box plot of the difference in transcription rate between A-labeled carRNA and unlabeled carRNA (inset). P values were determined using the Wilcoxon rank sum test. Only m 6 A-labeled repetitive RNA exhibits a significant left shift after Fto KO (indicating a significant reduction in transcription rate compared to unlabeled repetitive RNA). For (A) and (B), p values were determined using the Wilcoxon signed-rank test.

[0065] Figure 25 A to Figure 25 C. FTO-mediated m-associated with METTL3 and YTHDC1 6 Changes in A levels and carRNA expression. (25A) A difference in carRNA mRNA expression was observed between the hypermethylation after Fto KO and the hypomethylation after Mettl3 KO. 6 The negative correlation between the m6A level and the fold change of m6A level indicates that the m6A peak directly demethylated by Fto may be deposited by METTL3. (25B) There is a correlation between the m6A level of carRNA after hypomethylation after Fto KO and the m6A level after Mettl3 KO. 6 A positive correlation between the fold change of A level and the hypomethylation of m 6 Peak A may be caused by the blocked accessibility of METTL3 to its target. 6 A fold change in the level of carRNA and LINE1 RNA in each group was divided into 50 segments. The correlation between repeated RNA and LINE1 RNA is also shown in the scatter plot. (25C) A negative correlation between the transcript abundance fold change between carRNA downregulated after FtoKO and carRNA upregulated after Ythdc1 cKO was observed. Based on the fold change in transcript abundance after sorting Fto KO, carRNA and LINE1 RNA in each group were divided into 50 segments. For (A) to (C), r refers to the Pearson correlation coefficient. P values were calculated based on t distribution.

[0066] Figure 26 A to Figure 26L. Polyadenylated mRNA and snRNA are unlikely to be the main targets of FTO in mESC. (26A) shows the expression of polyadenylated mRNA and snRNA in WT mESC and FTO - / - ca-mRNA mRNA in mESCs 6 A horizontal box plot. (26B) shows WT mESC and Fto - / - mESCs exon and intron expression of ca-mRNA 6 A horizontal box plot. (26C) shows the differential methylation of ca-mRNA in mESCs after Fto KO. 6 Volcano plot of peak A. (26D) shows the hypermethylated mESCs 6 A-labeled ca-mRNA and other m 6 A. Box plots of the fold changes in expression of ca-mRNAs after Fto KO. P values were determined using the Wilcoxon rank sum test. For (A) to (D), the expression of chromatin-associated mRNAs (ca-mRNAs), most of which were pre-mRNAs, was evaluated. 6 A level and expression level. (26E and 26F) for WT mESC and Fto - / - mESC mRNA m 6 Quality analysis of AMeRIP-seq data. (E) 6 The top consensus motifs identified in peak A. (F) m from n = 2 replicates. 6 The overlap of peak A shows high reproducibility. (26G) shows the difference in mESCs after Fto KO. 6 A Volcano plot of methylation. (26H) by WT mESC and Mettl4 - / - mEB formation represented by cell proliferation of mESC after differentiation induction. (26I) WT mESC and Mettl4 - / - Proliferation of mESCs. (26J) Proliferation of mESCs from 3-week-old WT mice and Mettl4 - / - HE staining of mouse ovaries. Representative images were selected from 3 independent experiments. No obvious defects were observed after Mettl4 KO. (26K) - / - Comparable numbers of GV oocytes were collected from 9-week-old Mettl4 mice and WT controls. p values were determined using unpaired two-sided t-tests; error bars and mean ± SEM are shown for n = 6 mice. (26L) - / -Comparable numbers of SN and NSN GV oocytes were collected from mice. Error bars and mean ± SEM are shown for n = 3 mice. The SN / NSN ratio was similar to that of normal WT mice of this age. For (A) and (B), p values were determined using the Wilcoxon signed-rank test.

[0067] Figure 27 A to Figure 27 In Mel624 cells, FTO knockdown leads to more open chromatin and increased transcription through demethylation of mRNA encoding histone modifiers. (27A to 27C) mRNAs from human cell lines including HEK293T cells, GOS cells, HT29 cells, Jurkat cells, K562 cells, U2OS cells, U251 cells, and WPMY cells were analyzed. 6 AMeRIP-seq datasets, these datasets are from CRA001315 (13). Most of these cell lines are human cancer cell lines. (A) shows LINE1 RNA m 6 A scatter plot showing the negative correlation between LINE1 RNA expression and FTO expression. (B) A scatter plot showing no significant correlation between LINE1 RNA expression and FTO expression. (C) 6 A scatter plot showing no significant correlation between caRNA levels and expression levels. (27D) mRNA expression of caRNAs in control Mel624 cells and FTO-knockdown Mel624 cells after rRNA (left) and polyadenylated RNA (right) depletion detected by UHPLC-MS / MS. 6A / A percentage. (27E) Left: DNase I-TUNEL assay followed by fluorescence imaging showed that chromatin in FTO-knocked Mel624 cells was more open compared to control Mel624 cells. Right: Box plot showing relative TUNEL intensity. (27F) Left: EU labeling followed by fluorescence imaging showed that overall nascent RNA synthesis in FTO-knocked Mel624 cells was increased compared to control Mel624 cells. Right: Box plot showing relative EU intensity. For (E) and (F): Scale bar, 30 μm Cell nuclei were counterstained with DAPI. Representative images were selected from 3 independent experiments. EU intensity and TUNEL intensity were quantified in each cell nucleus by ImageJ. (27G) Relative LINE1 RNA expression in control Mel624 cells and FTO-knocked Mel624 cells quantified by RT-qPCR. (27H) Relative expression of total LINE1 RNA in control Mel624 cells and Mel624 cells knocked down with LINE1 ASOs, quantified by RT-qPCR. (27I) Knockdown of LINE1 RNA in Mel624 cells with LINE1 ASOs (red) resulted in slight changes in chromatin accessibility compared to the control ASO (grey), as detected by flow cytometry after a DNase I-TUNEL assay. (27J) Density plot of total ATAC-seq signal at the center of the peaks in control Mel624 cells and FTO knockdown Mel624 cells, quantified by RPKM. (27K) Volcano plot of differential ATAC peaks in FTO knockdown Mel624 cells compared to the control group is shown. Significantly increased peaks are shown in red (p < 0.01: 8097; FDR < 0.05: 261), and significantly decreased peaks are shown in blue (p < 0.01: 278; FDR < 0.05: 5). FeatureCounts and DESeq2 were used to determine the p-value and FDR value of the difference peak. The original peaks were merged into a joint peak set, and the significantly increased peaks obtained were more than the reduced peaks. (27L) The acquired open regions after FTO knockdown from ATAC-seq were enriched in the H3K27Ac regions identified from ChIP-seq. The ChIP-seq binding sites sorted by the acquired open regions from ATAC-seq were drawn in the heat map view. When comparing FTO knockdown with the control, it was observed that the H3K27Ac levels in these regions were higher. The acquired open regions were defined by a 1kb sliding window along the genome. (27M) Immunoblot analysis of the protein levels of H3K27Ac and H3K27Me3 in the Mel624 cells that FTO knocked down were dysregulated compared to the control.(27N) shows a heat map of dysregulated EP300, KDM6B, and EZH2 expression in FTO knockdown Mel624 cells compared to controls from mRNA-seq. (27O) shows m on EP300 transcripts, KDM6B transcripts, and EZH2 transcripts in FTO knockdown Mel624 cells. 6 A) IGV tracks with elevated peak levels compared to controls. (27P) Knockdown of YTHDF2 in shFTO Mel624 cells restored reduced EZH2 lifespan, while knockdown of IGF2BP1 to IGF2BP3 in shFTO Mel624 cells restored increased EP300 and KDM6B lifespans. For (D) to (H), p values were determined using an unpaired, two-sided t-test. For (D), (G), and (H), error bars and mean ± SD of n = 4 experiments are shown.

[0068] Figure 28 A to Figure 28 H. FTO regulates LINE1 RNA m 6 A, LINE1 RNA abundance and association of LINE1 RNA with DNA. (28A) Left: FISH showing reduced LINE1 RNA levels after Fto KO. Scale bar, 20 μm. Right: Quantification of WT mESC and Fto by ImageJ. - / - Box plot of relative FISH intensity in mESC. (28B) In WT mESC and Fto - / - In mESC, the relative m6A enrichment fold of total LINE1 RNA detected by MeRIP-qPCR (left), and the relative expression level of total LINE1 RNA detected by RT-qPCR (right). (28C) Top: shows the relative m6A enrichment fold of total LINE1 RNA detected by MeRIP-qPCR (left), and the relative expression level of total LINE1 RNA detected by RT-qPCR (right). - / - Immunoblot analysis of L1ORF1p in mESCs. Bottom: L1ORF1p levels relative to tubulin quantified by ImageJ. (28D) WT mESCs were treated with DMSO (as a control) or FTO inhibitor (FB23-2) for 72 hours. Left: Relative mRNA levels of total LINE1 RNA detected by MeRIP-qPCR. 6 A: Enrichment fold. Right: Relative expression of total LINE1 RNA detected by RT-qPCR. (28E) shows the relative expression of WT mESC and Fto - / -ChIRP-qPCR of relative DNA binding of LINE1 RNA in mESCs. Fto KO resulted in reduced DNA binding of total LINE1 RNA and selected Fto-targeted LINE1 subfamilies. Malat1 was used as a negative control. (28F) shows the relative DNA binding of WT mESCs and Fto - / - DRIP-qPCR of relative levels of LINE1 RNA:DNA hybrids in mESCs. Fto KO resulted in reduced R-loop formation for both total LINE1 RNA and selected FTO-targeted LINE1 subfamilies. The reduction in DNA association and R-loop formation is consistent with a decrease in LINE1 RNA transcription rate. (28G and 28H) For total LINE1 RNA and selected FTO-targeted LINE1 subfamilies, Fto KO resulted in reduced R-loop formation for both total LINE1 RNA and selected FTO-targeted LINE1 subfamilies compared to control gRNA. - / - Application of dCas13b-wtFTO and LINE1 gRNA in mESCs resulted in increased DNA binding (G) and restored R-loop formation (H).

[0069] Figure 29 A to Figure 29 C. Transient OE of Fto in zygotes increases the body weight of female F1 pups, but not male F1 pups. (29A) Schematic diagram of the experimental flow. (B and C) Body weights of female F1 pups (29B) and male F1 pups (29C) born from zygotes injected or not with Fto mRNA.

[0070] Figure 30 A to Figure 30 B. Overexpression of the Fto-GFP fusion protein in early embryos. (30A) Schematic diagram of the experimental flow. (30B) Fluorescence microscopy analysis of GFP signals in embryos with and without successful injection or translation of the Fto-GFP fusion mRNA. GFP-positive embryos can be selected for downstream analysis. Scale bar: 20 µm.

[0071] Figure 31 FTO OE increased the expression of MERVL and promoted its entry into the cell nucleus.

[0072] Figure 32 A to Figure 32H. Transient OE of FTO in zygotes increases implantation rate at E7.5 and increases embryo weight at E12.5. (32A) Development of FTO-control, FTO-KO, and FTO-OE embryos at E4.5. (32B) Average number of ICM and TE cells in FTO-control, FTO-KO, and FTO-OE embryos at E4.5. (32C) Imaging of the decidua and embryos of FTO-control and FTO-OE embryos at E7.5, related to the left panel of D. (32D) Calculation of embryo / decidua ratio at E7.5, BDF1 vs. BDF1 hybridization (left panel) and C57 vs. DBA2 hybridization (right panel). (32E) Embryonic and placental weights of FTO-control and FTO-OE embryos at E12.5. (32F) Placental development in FTO-control and FTO-OE groups at E12.5. Arrows point to the labyrinth layer. (32G) Weight tracking of FTO-control and FTO-OE pups from birth to adulthood. (32H) Glucose tolerance test performed on mice fed a normal diet and mice fed a high-fat diet (males only) at weeks 8 to 9. The high-fat diet was fed for 2 weeks before the test (n = 6 to 10). (32I) Representative photographs of FTO-control and FTO-OE mice at week 18 after 12 weeks of high-fat diet. (32J) Body weight of FTO-control and FTO-OE mice at week 18 after 12 weeks of high-fat diet. DETAILED DESCRIPTION

[0073] This article discloses RNA mRNAs that are helpful for mammalian development through FTO. 6 Functional substrates and mechanisms of A demethylation. This disclosure supports LINE1 RNA as the main substrate of FTO in mESC. Many aspects show that FTO also mediates the mRNA of other carRNAs. 6 In some cancer cells, FTO can be hijacked to mediate mRNA mRNA demethylation. 6 In contrast to FTO-mediated mESC demethylation (7–12), which may dominate the regulation of chromatin state (34) (Supplementary text and Fig. S22), our results suggest that FTO-mediated m 6 A demethylation maintains LINE1 RNA abundance. This may help promote local chromatin opening and activation of LINE1-containing genes. Aspects of this paper suggest that the FTO-LINE1 RNA axis has functional relevance in the development of mouse oocytes and embryos. 6In addition, RNA 5-methylcytosine oxidation is also known to affect the transcription of ERVL and ERVL-related genes in mESCs (35), suggesting that regulation by retrotransposon RNA modification may be widespread (36).

[0074] Also disclosed are FTO overexpression (transient or stable) for use in in vitro fertilization (IVF), artificial insemination, cloning, or improving natural fertilization and birth outcomes in mammals. Also included are IVF, artificial insemination, or improving natural fertilization in humans. Aspects related to artificial insemination can also be applied to sex-sorted semen (also known as sexed semen), a common method used in livestock reproduction.

[0075] In addition to humans, non-human mammals for which the FTO overexpression method can be used include, but are not limited to, livestock (domesticated agricultural animals), companion animals, endangered wild animals, and animals raised for research products or biomedical products.

[0076] A. Protein

[0077] Disclosed herein are FTO proteins and nucleic acids encoding such proteins. FTO proteins can be exogenous proteins introduced into cells to affect the RNA methylation pattern and development of the cells, as described herein.

[0078] As used herein, a "protein" or "polypeptide" refers to a molecule comprising at least five amino acid residues. In some aspects, the wild-type version of a protein or polypeptide is used. The above terms can be used interchangeably. A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, in particular its amino acid sequence, is altered relative to the wild-type protein or polypeptide. In some aspects, the modified / variant protein or polypeptide has at least one modified activity or function (it should be recognized that a protein or polypeptide can have multiple activities or functions). It is particularly disclosed that a modified / variant protein or polypeptide can be altered in one activity or function but retain the activity or function of the wild-type in other aspects, such as enzymatic activity.

[0079] Proteins, including FTO proteins, can be isolated directly from their naturally occurring organisms, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In a specific aspect, there are isolated nucleic acid fragments and recombinant vectors that incorporate nucleic acid sequences encoding polypeptides (e.g., FTO polypeptides or fragments thereof). The term "recombinant" can be used in conjunction with the name of a polypeptide or a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been processed in vitro or as a replication product of such a molecule.

[0080] In certain aspects, the size of the FTO protein or polypeptide (wild type or modified) can include, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more than 2500 amino acid residues, and any range derived therefrom, or the derivative range of the corresponding amino acid sequence described or cited herein. It is disclosed that the polypeptides, including the FTO polypeptides used herein, can be mutated by truncation to make them shorter than their corresponding wild-type forms, and they can be changed by fusion or conjugation with heterologous protein sequences or heterologous polypeptide sequences having specific functions (e.g., for targeting or positioning, for enhancing immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any different functional unit or structural unit of a protein or polypeptide, and generally refers to an amino acid sequence having a structure or function that can be recognized by those skilled in the art.

[0081] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 250, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 (or any range derivable therefrom) or more than 50 variant amino acid or nucleic acid substitutions, or having a 60%, 61%, 62%, 63%, 64%, 65%, 66%, or similar amino acid substitution(s) therewith. %, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivative therefrom) similarity, identity or homology.

[0082] 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 1 16, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、 166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、 241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、 266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、 341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、 366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、 391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、 441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、 466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、 491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、 591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、 666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、 766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815,816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862 2, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908 , 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955 or 1000 amino acids.

[0083] 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 ,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115, 116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、 141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、 166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、 241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、 266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、 341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、 366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、 391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、 441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、 466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、 491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、 591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、 666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763、764、765、 766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、789、790、 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815,816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863 63, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910 , 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957 990, 991, 992, 993, 994, 995, 996, 997, 998, 999 or 1000 (or any range derivable therefrom) consecutive amino acids.

[0084] 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613,614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、779、780、781、782、783、784、785、786、787、788、 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813,814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838 , 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863 , 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888 , 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913 , 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938 , 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963 , 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999 or 1000 (or ranges derivable therefrom) consecutive amino acids that share at least, at most or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% similarity, identity or homology.

[0085] In some aspects, there is a nucleic acid or polypeptide that is present at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65 46、Position 47、Position 48、Position 49、Position 50、Position 51、Position 52、Position 53、Position 54、Position 55、Position 56、Position 57、Position 58、Position 59、Position 60、Position 61、Position 62、Position 63、Position 64、Position 65、Position 66、Position 67、Position 68、Position 69、Position 70、Position 71、Position 72、Position 73、Position 74、Position 75、Position 76、Position 77、Position 78、Position 79、Position 80、Position 81、Position 82、Position 83、Position 84、Position 85、Position 86、Position 87、Position 88、Position 89、Position 90、Position 91、Position 92、Position 93、Position 94、Position 95、Position Position 96, Position 97, Position 98, Position 99, Position 100, Position 101, Position 102, Position 103, Position 104, Position 105, Position 106, Position 107, Position 108, Position 109, Position 110, Position 111, Position 112, Position 113, Position 114, Position 115, Position 116, Position 117, Position 118, Position 119, Position 120, Position 121, Position 122, Position 123, Position 124, Position 125, Position 126, Position 127, Position 128, Position 129, Position 130, Position 131, Position 132, Position 133, Position 134, Position 135, Position 136, Position 137, Position 138、Position 139、Position 140、Position 141、Position 142、Position 143、Position 144、Position 145、Position 146、Position 147、Position 148、Position 149、Position 150、Position 151、Position 152、Position 153、Position 154、Position 155、Position 156、Position 157、Position 158、Position 159、Position 160、Position 161、Position 162、Position 163、Position 164、Position 165、Position 166、Position 167、Position 168、Position 169、Position 170、Position 171、Position 172、Position 173、Position 174、Position 175、Position 176、Position 177、Position 178、Position 179、Position 180, Position 181, Position 182, Position 183, Position 184, Position 185, Position 186, Position 187, Position 188, Position 189, Position 190, Position 191, Position 192, Position 193, Position 194, Position 195, Position 196, Position 197, Position 198, Position 199, Position 200, Position 201, Position 202, Position 203, Position 204, Position 205, Position 206, Position 207, Position 208, Position 209, Position 210, Position 211, Position 212, Position 213, Position 214, Position 215, Position 216, Position 217, Position 218, Position 219, Position 220, Position 221 21. Position 222, position 223, position 224, position 225, position 226, position 227, position 228, position 229, position 230, position 231, position 232, position 233, position 234, position 235, position 236, position 237, position 238, position 239, position 240, position 241, position 242, position 243, position 244, position 245, position 246, position 247, position 248, position 249, position 250, position 251, position 252, position 253, position 254, position 255, position 256, position 257, position 258, position 259, position 260, position 261, position 262, Position 263, Position 264, Position 265, Position 266, Position 267, Position 268, Position 269, Position 270, Position 271, Position 272, Position 273, Position 274, Position 275, Position 276, Position 277, Position 278, Position 279, Position 280, Position 281, Position 282, Position 283, Position 284, Position 285, Position 286, Position 287, Position 288, Position 289, Position 290, Position 291, Position 292, Position 293, Position 294, Position 295, Position 296, Position 297, Position 298, Position 299, Position 300, Position 301, Position 302, Position 303, Position 304 04, position 305, position 306, position 307, position 308, position 309, position 310, position 311, position 312, position 313, position 314, position 315, position 316, position 317, position 318, position 319, position 320, position 321, position 322, position 323, position 324, position 325, position 326, position 327, position 328, position 329, position 330, position 331, position 332, position 333, position 334, position 335, position 336, position 337, position 338, position 339, position 340, position 341, position 342, position 343, position 344, position 345,Position 346, position 347, position 348, position 349, position 350, position 351, position 352, position 353, position 354, position 355, position 356, position 357, position 358, position 359, position 360, position 361, position 362, position 363, position 364, position 365, position 366, position 367, position 368, position 369, position 370, position 371, position 372, position 373, position 374, position 375, position 376, position 377, position 378, position 379, position 380, position 381, position 382, position 383, position 384, position 385, position 386, position 387 87, position 388, position 389, position 390, position 391, position 392, position 393, position 394, position 395, position 396, position 397, position 398, position 399, position 400, position 401, position 402, position 403, position 404, position 405, position 406, position 407, position 408, position 409, position 410, position 411, position 412, position 413, position 414, position 415, position 416, position 417, position 418, position 419, position 420, position 421, position 422, position 423, position 424, position 425, position 426, position 427, position 428, Position 429, Position 430, Position 431, Position 432, Position 433, Position 434, Position 435, Position 436, Position 437, Position 438, Position 439, Position 440, Position 441, Position 442, Position 443, Position 444, Position 445, Position 446, Position 447, Position 448, Position 449, Position 450, Position 451, Position 452, Position 453, Position 454, Position 455, Position 456, Position 457, Position 458, Position 459, Position 460, Position 461, Position 462, Position 463, Position 464, Position 465, Position 466, Position 467, Position 468, Position 469, Position 470 70, position 471, position 472, position 473, position 474, position 475, position 476, position 477, position 478, position 479, position 480, position 481, position 482, position 483, position 484, position 485, position 486, position 487, position 488, position 489, position 490, position 491, position 492, position 493, position 494, position 495, position 496, position 497, position 498, position 499, position 500, position 501, position 502, position 503, position 504, position 505, position 506, position 507, position 508, position 509, position 510, position 511,Position 512, position 513, position 514, position 515, position 516, position 517, position 518, position 519, position 520, position 521, position 522, position 523, position 524, position 525, position 526, position 527, position 528, position 529, position 530, position 531, position 532, position 533, position 534, position 535, position 536, position 537, position 538, position 539, position 540, position 541, position 542, position 543, position 544, position 545, position 546, position 547, position 548, position 549, position 550, position 551, position 552, position 553 53, position 554, position 555, position 556, position 557, position 558, position 559, position 560, position 561, position 562, position 563, position 564, position 565, position 566, position 567, position 568, position 569, position 570, position 571, position 572, position 573, position 574, position 575, position 576, position 577, position 578, position 579, position 580, position 581, position 582, position 583, position 584, position 585, position 586, position 587, position 588, position 589, position 590, position 591, position 592, position 593, position 594, Position 595, position 596, position 597, position 598, position 599, position 600, position 601, position 602, position 603, position 604, position 605, position 606, position 607, position 608, position 609, position 610, position 611, position 612, position 613, position 614, position 615, position 616, position 617, position 618, position 619, position 620, position 621, position 622, position 623, position 624, position 625, position 626, position 627, position 628, position 629, position 630, position 631, position 632, position 633, position 634, position 635, position 636 36. Position 637, position 638, position 639, position 640, position 641, position 642, position 643, position 644, position 645, position 646, position 647, position 648, position 649, position 650, position 651, position 652, position 653, position 654, position 655, position 656, position 657, position 658, position 659, position 660, position 661, position 662, position 663, position 664, position 665, position 666, position 667, position 668, position 669, position 670, position 671, position 672, position 673, position 674, position 675, position 676, position 677,Position 678, position 679, position 680, position 681, position 682, position 683, position 684, position 685, position 686, position 687, position 688, position 689, position 690, position 691, position 692, position 693, position 694, position 695, position 696, position 697, position 698, position 699, position 700, position 701, position 702, position 703, position 704, position 705, position 706, position 707, position 708, position 709, position 710, position 711, position 712, position 713, position 714, position 715, position 716, position 717, position 718, position 719 19. Position 720, position 721, position 722, position 723, position 724, position 725, position 726, position 727, position 728, position 729, position 730, position 731, position 732, position 733, position 734, position 735, position 736, position 737, position 738, position 739, position 740, position 741, position 742, position 743, position 744, position 745, position 746, position 747, position 748, position 749, position 750, position 751, position 752, position 753, position 754, position 755, position 756, position 757, position 758, position 759, position 760, Position 761, position 762, position 763, position 764, position 765, position 766, position 767, position 768, position 769, position 770, position 771, position 772, position 773, position 774, position 775, position 776, position 777, position 778, position 779, position 780, position 781, position 782, position 783, position 784, position 785, position 786, position 787, position 788, position 789, position 790, position 791, position 792, position 793, position 794, position 795, position 796, position 797, position 798, position 799, position 800, position 801, position 802 02, position 803, position 804, position 805, position 806, position 807, position 808, position 809, position 810, position 811, position 812, position 813, position 814, position 815, position 816, position 817, position 818, position 819, position 820, position 821, position 822, position 823, position 824, position 825, position 826, position 827, position 828, position 829, position 830, position 831, position 832, position 833, position 834, position 835, position 836, position 837, position 838, position 839, position 840, position 841, position 842, position 843,Position 844, position 845, position 846, position 847, position 848, position 849, position 850, position 851, position 852, position 853, position 854, position 855, position 856, position 857, position 858, position 859, position 860, position 861, position 862, position 863, position 864, position 865, position 866, position 867, position 868, position 869, position 870, position 871, position 872, position 873, position 874, position 875, position 876, position 877, position 878, position 879, position 880, position 881, position 882, position 883, position 884, position 885 5. Position 886, position 887, position 888, position 889, position 890, position 891, position 892, position 893, position 894, position 895, position 896, position 897, position 898, position 899, position 900, position 901, position 902, position 903, position 904, position 905, position 906, position 907, position 908, position 909, position 910, position 911, position 912, position 913, position 914, position 915, position 916, position 917, position 918, position 919, position 920, position 921, position 922, position 923, position 924, position 925, position 926, position 927, position 928, position 929, position 930, position 931, position 932, position 933, position 934, position 935, position 936, position 937, position 938, position 939, position 940, position 941, position 942, position 943, position 944, position 945, position 946, position 947, position 948, position 949, position 950, position 951, position 952, position 953, position 954, position 955, position 956, position 957, position 958, position 959, position 960, position 961, position 962, position 963, position 964, position 965, position 966, position 967, position 968, 994, position 995, position 996, position 997, position 998, position 999, or position 1000, and comprises at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of any of the gene products identified in Table 1.11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 , 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 , 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 , 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628,629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754、755、756、757、758、759、760、761、762、763、764、765、766、767、768、769、770、771、772、773、774、775、776、777、778、 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828,829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872 , 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916 16, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959 or 1000 consecutive amino acids or consecutive nucleotides.

[0086] 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 70, position 71, position 72, position 73, position 74, position 75, position 76, position 77, position 78, position 79, position 80, position 81, position 82, position 83, position 84, position 85 Position 49, Position 50, Position 51, Position 52, Position 53, Position 54, Position 55, Position 56, Position 57, Position 58, Position 59, Position 60, Position 61, Position 62, Position 63, Position 64, Position 65, Position 66, Position 67, Position 68, Position 69, Position 70, Position 71, Position 72, Position 73, Position 74, Position 75, Position 76, Position 77, Position 78, Position 79, Position 80, Position 81, Position 82, Position 83, Position 84, Position 85, Position 86, Position 87, Position 88, Position 89, Position 90, Position 91, Position 92, Position 93, Position 94, Position 95, Position 96, Position 97, Position 98 , Position 99, Position 100, Position 101, Position 102, Position 103, Position 104, Position 105, Position 106, Position 107, Position 108, Position 109, Position 110, Position 111, Position 112, Position 113, Position 114, Position 115, Position 116, Position 117, Position 118, Position 119, Position 120, Position 121, Position 122, Position 123, Position 124, Position 125, Position 126, Position 127, Position 128, Position 129, Position 130, Position 131, Position 132, Position 133, Position 134, Position 135, Position 136, Position 137, Position 138, Position 139, Position 140 40, position 141, position 142, position 143, position 144, position 145, position 146, position 147, position 148, position 149, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 163, position 164, position 165, position 166, position 167, position 168, position 169, position 170, position 171, position 172, position 173, position 174, position 175, position 176, position 177, position 178, position 179, position 180, position 181,A polypeptide or a nucleic acid encoding a gene product identified in Table 1 with substitution, deletion or insertion of one or more positions including position 182, position 183, position 184, position 185, position 186, position 187, position 188, position 189, position 190, position 191, position 192, position 193, position 194, position 195, position 196, position 197, position 198, position 199, position 200 (or any range derivable therefrom), as long as the polypeptide has an FTO activity equivalent to at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130% of that of the unsubstituted protein or wild-type FTO protein.

[0087] Disclosed herein are uses of FTO proteins. The FTO protein used can be any FTO protein homolog that can cause the desired outcome, including improvement in in vitro fertilization (such as increasing implantation rate). The FTO protein used can be from any organism. In some aspects, the FTO protein sequence is from an animal. In some aspects, the FTO protein sequence is from a mammal. In some aspects, the FTO protein sequence is from a rodent. In some aspects, the FTO protein sequence is from a livestock animal. In some aspects, the FTO protein sequence is from a companion animal. In some aspects, the FTO sequence is from a wild animal. In some aspects, the FTO protein sequence is from a primate. In some aspects, the FTO protein sequence is from a human. In some aspects, the FTO protein sequence is from a starfish. In some aspects, the FTO protein sequence is from the species listed in Table 1.

[0088] Table 1: Identifiers of FTO sequences, all of which are incorporated herein by reference

[0089] Accession number (species) NM_001080432.3 (Homo sapiens – human) XM_015126148.2 (Macaca mulatta - rhesus monkey) NM_011936.2 (Mus musculus – mouse) NM_001039713.1 (Rattus norvegicus – Rat) NM_001098142.1 (Domestic cattle (Bos taurus) – cattle) XM_019979409.1 (Domestic Cattle (Bos taurus) – Cattle) XM_005890937.1 (Domestic Cattle (Bos taurus) – Cattle) NM_001112692.1 (Eurasian wild boar (Sus scrofa) – pig) XM_038659338.1 (Dog (Canis Lupis familiaris) – Dog) NM_001104931.1 (Domestic sheep (Ovis aries) – Sheep) XP_022097537.1 (Crown-of-thorns starfish (Acanthaster planci) – starfish) XP_038078604 (Patiria miniate - starfish) XP_033625687.1 (Red Sea Starfish (Asterias rubens) - Starfish) ALR88588.1 (Saccoglossus kowalevskii – Acorn beetle) XP_001420808.1 (Ostreococcus lucimarinus) XM_009303351.3 (Danio rerio – Zebrafish) XP_022840077.1 (Ostreococcus tauri) OUS43030.1 (Ostreococcus tauri) CAD8812083.1 (Ostreococcus mediterraneus) XP_002502764.1 (Micromonas commode) XP_003060875.1 (Micromonas pusilla) CAD8435953.1 (Micromonas pusilla) CAE3374532.1 (Micromonas Polaris)

[0090] The FTO protein and / or nucleic acid encoding the FTO protein introduced or provided to the cells of the present disclosure can be a sequence corresponding to the species of the cell. For example, an FTO protein comprising a human FTO sequence can be introduced into a human cell. Similarly, a nucleic acid encoding a human FTO sequence can be introduced into a human cell.

[0091] The nucleotide and protein, polypeptide and peptide sequences of various genes have been previously disclosed and can be found in recognized computer databases. Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (global website: www.ncbi.nlm.nih.gov / ) and the Universal Protein Resource Database (UniProt; global website: www.uniprot.org / ). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or techniques known to those of ordinary skill in the art.

[0092] It is disclosed that the compositions of the present disclosure contain from about 0.001 mg to about 10 mg of total polypeptides, peptides and / or proteins per mL. The protein concentration in the composition can be about, at least about, or at most about 0.001 ng / mL or 0.001 mg / mL, 0.010 ng / mL or 0.010 mg / mL, 0.050 ng / mL or 0.050 mg / mL, 0.1 ng / mL or 0.1 mg / mL, 0.2 ng / mL or 0.2 mg / mL, 0.3 ng / mL or 0.3 mg / mL, 0.4 ng / mL or 0.4 mg / mL, 0.5 ng / mL or 0.5 mg / mL, 0.6 ng / mL or 0.6 mg / mL, 0.7 ng / mL or 0.7 mg / mL, 0.8 ng / mL or 0.8 mg / mL, 0.9 ng / mL or 0.9 mg / mL, 1.0 ng / mL or 1.0 mg / mL, 1.5 ng / mL or 1.5 mg / mL, 2.0 ng / mL or 2.0 mg / mL, 2.5 ng / mL or 2.5 mg / mL, 10.0 ng / mL or more (or any range derivable therein).

[0093] 1. Variant polypeptides

[0094] The following discusses altering the amino acid subunits of proteins, including FTO proteins, to create equivalent or even improved second-generation variant polypeptides or peptides. For example, certain amino acids can replace other amino acids in a protein or polypeptide sequence with or without significant loss of enzymatic activity. Since the ability and properties of a protein to interact determine the functional activity of the protein, certain amino acid substitutions can be made in a protein sequence and its corresponding DNA coding sequence while still producing a protein with similar or desired properties. Therefore, the present inventors disclose that various changes can be made to the DNA sequence of a gene encoding a protein without significantly losing its biological utility or activity.

[0095] As used herein, the term "functionally equivalent codons" refers to codons that encode the same amino acid, such as the six different codons that encode arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to changes in one or more than one codon that encode a biologically equivalent amino acid.

[0096] The amino acid sequence variants of the present invention can be substitution variants, insertion variants or deletion variants. Compared to the wild type, the variation of the polypeptide of the present invention can affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more than 50 discontinuous or continuous amino acids of the protein or polypeptide. Variants can include amino acid sequences having at least 50%, 60%, 70%, 80% or 90% identity to any of the sequences provided or referenced herein, including all values and ranges therebetween. Variants can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 20 substituted amino acids....

Claims

1. A method for increasing zygote implantation in an animal, comprising implanting one or more fertilized cells into the reproductive tract of the animal, wherein prior to implantation of the fertilized cells, exogenous fat mass and obesity-related protein (FTO) and / or a nucleic acid molecule encoding FTO capable of transiently overexpressing exogenous FTO are introduced into the fertilized cells.

2. The method of claim 1, wherein the fertilized cells are implanted into the uterus or endometrium of an animal.

3. The method according to claim 1 or 2, wherein the fertilized cell is in the zygote development stage when the nucleic acid and / or protein is introduced.

4. The method according to any one of claims 1 to 3, wherein implantation occurs when the fertilized cells are at the blastocyst stage of development.

5. The method according to any one of claims 1 to 4, wherein the fertilized cells are contacted with the nucleic acid by injecting the nucleic acid into the fertilized cells. 6 . The method according to claim 1 , wherein exogenous FTO cannot be detected in the fertilized cells when the fertilized cells are in the embryonic development stage.

7. The method according to any one of claims 1 to 6, wherein the fertilized cells are fertilized in vitro.

8. The method according to any one of claims 1 to 6, wherein the fertilized cells are produced by natural mating or artificial insemination.

9. The method according to any one of claims 1 to 8, wherein the fertilized cells are collected from the animal by non-surgical techniques or surgical techniques.

10. The method according to any one of claims 1 to 9, wherein the animal is a livestock animal.

11. The method according to any one of claims 1 to 10, wherein the animal is a cow.

12. The method according to any one of claims 1 to 10, wherein the animal is a pig.

13. The method of any one of claims 1 to 9, wherein the animal is a companion animal.

14. The method of claim 13, wherein the companion animal is a dog or a cat.

15. The method of any one of claims 1 to 9, wherein the animal is a research animal or an animal used for xenotransplantation.

16. The method of claim 15, wherein the research animal is a mouse or a rat.

17. The method of any one of claims 1 to 9, wherein the animal is a human.

18. The method of any one of claims 1 to 9, wherein the animal is an endangered animal.

19. The method of any one of claims 1 to 17, wherein the animal is of a different species than the species of the fertilized cell.

20. The method according to any one of claims 1 to 19, wherein the nucleic acid molecule encoding FTO is an mRNA molecule.

21. The method according to any one of claims 1 to 19, wherein the nucleic acid molecule encoding FTO is a DNA molecule.

22. The method of claim 21, wherein the DNA molecule is an expression construct.

23. The method according to any one of claims 1 to 22, wherein a viral vector is used, and the nucleic acid molecule is introduced into the fertilized cell by using the viral vector.

24. The method according to any one of claims 1 to 22, wherein the nucleic acid molecule is introduced into the fertilized cell by transfection.

25. The method of any one of claims 1 to 24, wherein the method further comprises monitoring the implantation of the fertilized cells in the animal.

26. The method of any one of claims 1 to 25, wherein the method further comprises monitoring the pregnancy of the animal.

27. The method of any one of claims 1 to 26, wherein the method further comprises detecting one or more than one pregnancy biomarker in the animal.

28. The method of claim 27, wherein the pregnancy biomarker comprises human chorionic gonadotropin.

29. The method of any one of claims 1 to 28, wherein the method further comprises detecting the demethylation status in the fertilized cells.

30. The method according to any one of claims 1 to 29, wherein the method further comprises culturing and / or incubating the cells in culture medium and / or buffer.

31. A method for increasing the birth weight of an animal's offspring, comprising implanting one or more fertilized cells into the reproductive tract of the animal, wherein exogenous fat mass and obesity-related protein (FTO) and / or a nucleic acid molecule encoding FTO capable of transiently overexpressing exogenous FTO are introduced into the fertilized cells prior to implantation of the fertilized cells.

32. The method of claim 31 , wherein the fertilized cells are implanted into the uterus or endometrium of an animal.

33. The method of claim 31 or 32, wherein the fertilized cell is at the zygote stage of development when the nucleic acid and / or protein is introduced.

34. The method of any one of claims 31 to 33, wherein implantation occurs when the fertilized cells are at the blastocyst stage of development.

35. The method of any one of claims 31 to 34, wherein the fertilized cell is contacted with the nucleic acid by injecting the nucleic acid into the fertilized cell.

36. The method according to any one of claims 31 to 35, wherein exogenous FTO is undetectable in the fertilized cells when the fertilized cells are in the embryonic development stage.

37. The method of any one of claims 31 to 36, wherein the fertilized cells are fertilized in vitro.

38. The method according to any one of claims 31 to 36, wherein the fertilized cells are produced by natural mating or artificial insemination.

39. The method of any one of claims 31 to 38, wherein the fertilized cells are collected from the animal by non-surgical or surgical techniques.

40. The method of any one of claims 31 to 39, wherein the animal is a livestock animal.

41. The method of any one of claims 31 to 40, wherein the animal is a cow.

42. The method of any one of claims 31 to 40, wherein the animal is a pig.

43. The method of any one of claims 31 to 39, wherein the animal is a companion animal.

44. The method of claim 43, wherein the companion animal is a dog or a cat.

45. The method of any one of claims 31 to 39, wherein the animal is a research animal or an animal for xenotransplant production.

46. The method of claim 45, wherein the study animal is a mouse or a rat.

47. The method of any one of claims 31 to 39, wherein the animal is a human.

48. The method of any one of claims 31 to 39, wherein the animal is an endangered animal.

49. The method of any one of claims 31 to 47, wherein the animal is of a different species than the species of the fertilized cell.

50. The method of any one of claims 31 to 49, wherein the nucleic acid molecule encoding FTO is an mRNA molecule.

51. The method of any one of claims 31 to 49, wherein the nucleic acid molecule encoding FTO is a DNA molecule.

52. The method of claim 51, wherein the DNA molecule is an expression construct.

53. The method according to any one of claims 31 to 52, wherein a viral vector is used and the nucleic acid molecule is introduced into the fertilized cell by using the viral vector.

54. The method of any one of claims 31 to 52, wherein the nucleic acid molecule is introduced into the fertilized cell by transfection.

55. The method of any one of claims 31 to 54, wherein the method further comprises monitoring the implantation of the fertilized cells in the animal.

56. The method of any one of claims 31 to 55, wherein the method further comprises monitoring the animal for pregnancy.

57. The method of any one of claims 31 to 56, wherein the method further comprises detecting one or more than one pregnancy biomarker in the animal.

58. The method of claim 57, wherein the pregnancy biomarker comprises human chorionic gonadotropin.

59. The method of any one of claims 31 to 58, wherein the method further comprises detecting the demethylation status in the fertilized cells.

60. The method according to any one of claims 31 to 59, wherein the method further comprises culturing and / or incubating the cells in culture medium and / or buffer.

61. A method for increasing the birth weight of an animal's offspring, comprising implanting one or more fertilized cells into the reproductive tract of the animal, wherein exogenous fat mass and obesity-related protein (FTO) and / or a nucleic acid molecule encoding FTO capable of transiently overexpressing exogenous FTO are introduced into the fertilized cells prior to implantation of the fertilized cells.

62. The method of claim 61, wherein the fertilized cells are implanted into the uterus or endometrium of an animal.

63. The method of claim 61 or 62, wherein the fertilized cell is at the zygote stage of development when the nucleic acid and / or protein is introduced.

64. The method of any one of claims 61 to 63, wherein implantation occurs when the fertilized cells are at the blastocyst stage of development.

65. The method of any one of claims 61 to 64, wherein the fertilized cell is contacted with the nucleic acid by injecting the nucleic acid into the fertilized cell.

66. The method according to any one of claims 61 to 65, wherein exogenous FTO is undetectable in the fertilized cell when the fertilized cell is in the embryonic development stage.

67. The method of any one of claims 61 to 66, wherein the fertilized cells are fertilized in vitro.

68. The method of any one of claims 61 to 66, wherein the fertilized cells are produced by natural mating or artificial insemination.

69. The method of any one of claims 61 to 68, wherein the fertilized cells are collected from the animal by non-surgical or surgical techniques.

70. The method of any one of claims 61 to 69, wherein the animal is a livestock animal.

71. The method of any one of claims 61 to 70, wherein the animal is a cattle.

72. The method of any one of claims 61 to 70, wherein the animal is a pig.

73. The method of any one of claims 61 to 69, wherein the animal is a companion animal.

74. The method of claim 73, wherein the companion animal is a dog or a cat.

75. The method of any one of claims 61 to 69, wherein the animal is a research animal or an animal for xenotransplant production.

76. The method of claim 75, wherein the study animal is a mouse or a rat.

77. The method of any one of claims 61 to 69, wherein the animal is a human.

78. The method of any one of claims 61 to 69, wherein the animal is an endangered animal.

79. The method of any one of claims 61 to 77, wherein the animal is of a species different from that of the fertilized cell.

80. The method of any one of claims 61 to 79, wherein the nucleic acid molecule encoding FTO is an mRNA molecule.

81. The method of any one of claims 61 to 79, wherein the nucleic acid molecule encoding FTO is a DNA molecule.

82. The method of claim 81, wherein the DNA molecule is an expression construct.

83. The method according to any one of claims 61 to 82, wherein a viral vector is used and the nucleic acid molecule is introduced into the fertilized cell by using the viral vector.

84. The method of any one of claims 61 to 82, wherein the nucleic acid molecule is introduced into the fertilized cell by transfection.

85. The method of any one of claims 61 to 84, wherein the method further comprises monitoring the implantation of fertilized cells in an animal.

86. The method of any one of claims 61 to 85, wherein the method further comprises monitoring the animal for pregnancy.

87. The method of any one of claims 61 to 86, wherein the method further comprises detecting one or more than one pregnancy biomarker in the animal.

88. The method of claim 87, wherein the pregnancy biomarker comprises human chorionic gonadotropin.

89. The method of any one of claims 61 to 88, wherein the method further comprises detecting the demethylation state in the fertilized cells.

90. The method of any one of claims 61 to 89, wherein the method further comprises culturing and / or incubating the cells in culture medium and / or buffer.

91. A fertilized cell comprising exogenous FTO nucleic acid and / or exogenous FTO protein.

92. The fertilized cell of claim 91, wherein the fertilized cell is in the zygote stage of development.

93. The fertilized cell of claim 91 or 92, wherein the fertilized cell is a human cell, a livestock animal cell, a companion animal cell, or a research animal cell.

94. The fertilized cell of any one of claims 92 to 93, wherein the fertilized cell is produced using the method of any one of claims 5 to 25.

95. A composition comprising the fertilized cell of any one of claims 91 to 94.

96. A composition comprising the fertilized cell of any one of claims 91 to 94 and one or more reagents for in vitro fertilization.

97. A method for demethylating long interspersed element-1 (LINE1) RNA in a cell, wherein the method comprises providing an effective amount of fat mass and obesity-associated protein (FTO) and / or a nucleic acid molecule encoding FTO to the cell.

98. The method of claim 97, wherein LINE1 RNA is a chromatin-associated regulatory RNA.

99. The method of claim 97 or 98, wherein the method comprises providing a nucleic acid encoding FTO.

100. The method of claim 97 or 98, wherein the method comprises providing an FTO protein.

101. The method of any one of claims 97 to 100, wherein methylation is m 6 A methylation.

102. A method of altering gene expression levels in a cell, wherein the method comprises providing an effective amount of FTO or a nucleic acid molecule encoding FTO to the cell.

103. The method of claim 102, wherein the method comprises providing a nucleic acid encoding FTO.

104. The method of claim 102, wherein the method comprises providing an FTO protein.

105. The method of any one of claims 102 to 104, wherein the gene comprises a LINE1 element.

106. The method of any one of claims 102 to 104, wherein the gene is a 2C gene.

107. A method of increasing chromatin accessibility in a cell, wherein the method comprises providing an effective amount of FTO or a nucleic acid molecule encoding FTO to the cell.

108. The method of claim 107, wherein the amount is effective to reduce the amount of histone modification in the cell.

109. The method of claim 107 or 108, wherein the method comprises providing a nucleic acid encoding FTO.

110. The method of claim 107 or 108, wherein the method comprises providing an FTO protein.

111. The method of any one of claims 97 to 110, wherein the cells are stem cells.

112. The method of claim 111, wherein the cells are embryonic stem cells.

113. The method of claim 111, wherein the cells are induced pluripotent stem cells.

114. The method of any one of claims 97 to 113, wherein the cell is a mouse cell.

115. The method of any one of claims 97 to 113, wherein the cell is a human cell.

116. The method of any one of claims 97 to 115, wherein the cell is a cancer cell.

117. A method for optimizing germ cell development, the method comprising providing an effective amount of FTO or a nucleic acid molecule encoding FTO to a cell.

118. The method of claim 117, wherein the amount is effective to increase chromatin accessibility in the cell.

119. The method of claim 117 or 118, wherein the method comprises providing a nucleic acid encoding FTO.

120. The method of claim 117 or 118, wherein the method comprises providing an FTO protein.

121. The method of any one of claims 117 to 120, wherein the germ cell is an oocyte.

122. The method of any one of claims 117 to 121, wherein the germ cells are mouse cells.

123. The method of any one of claims 97 to 122, wherein the method further comprises detecting the methylation status in the cell.

124. The method of any one of claims 97 to 123, wherein the method further comprises culturing the cells.

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