Methods and uses of transgenic mouse lines

Abstract

Disclosed herein, in certain embodiments, is a transgenic mouse that expresses a fusion protein that includes OCT4 under transcriptional control.In some embodiments, also disclosed herein includes embryos, stem cells, and germline cells obtained from the transgenic mouse.In additional embodiments, disclosed herein includes a method for generating a transgenic mouse, and a method for evaluating the product using embryos obtained from the transgenic mouse.

Description

[Technical field]

[0001] background This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 213,335, filed June 22, 2021, which is incorporated by reference in its entirety.

[0002] Genetically engineered mouse models are important for studying the function of genes at the whole animal level. Gene knockout mice, which represent gene loss-of-function strategies, and transgenic mice, which represent gain-of-function approaches, can be utilized to assess the molecular and cellular functions of genes or proteins of interest. Generally, transgenic mice can be generated by microinjecting transgenic constructs into fertilized eggs (oocytes or zygotes). Alternatively, retroviral vectors containing the transgene may be introduced into the eggs for subsequent generation of transgenic mice.

[0003] During development, the preimplantation embryo rapidly transforms from a metabolically quiescent, undifferentiated single cell under genetic control of maternal transcripts to a dynamic, multicellular embryo with a developed homeostatic machinery and its own functioning genome in just a few days (Leese 1991; Lane 2001; Gardner et al. 2005). The early embryo relies on pyruvate-based metabolism and relies solely on mitochondrial oxidative phosphorylation for energy production; like single-celled organisms, the early embryo lacks many key regulatory functions for pH and osmoregulation. After compaction to the 8-16 cell stage, there is a shift in metabolic control to a highly glycolytic metabolism. At the same time, there is a significant transition in the functional complexity of other cellular mechanisms as the physiology of the embryo becomes more similar to that of the somatic cell. It is the raw nature of early homeostatic regulation in the early embryo and subsequent development through the later stages of preimplantation development that poses serious problems in the laboratory. Maintenance of a favorable in vitro environment, particularly with modulation of one or more genes or proteins of interest, is essential to maximize survival and promote ongoing development.

[0004] Perturbations to the environment surrounding developing embryos in culture relative to the "normal" conditions encountered in the reproductive tract result in reduced embryo survival and impaired development. Thus, there is a need for sensitive and reproducible methods and assays to assess embryo development and toxicity.

[0005] Summary of the Invention In certain embodiments, disclosed herein is a transgenic mouse that expresses a fusion protein that includes OCT4 under transcriptional control.In some embodiments, also disclosed herein includes embryos, stem cells, and germline cells obtained from the transgenic mouse.In additional embodiments, disclosed herein includes a method for generating a transgenic mouse, and a method for evaluating the product using embryos obtained from the transgenic mouse.

[0006] In some embodiments, disclosed herein is a transgenic mouse comprising a stable expression of a fusion protein comprising octamer-binding transcription factor 4 (OCT4) under transcriptional control. In some cases, the gene expression of the fusion protein is stably transmitted through germline DNA. In some cases, an embryo expressing the OCT4::EGFP fusion protein can be generated, in which an oocyte is fertilized with sperm comprising the OCT4::EGFP fusion protein, and the sperm is derived from the transgenic mouse. In some cases, stem cells expressing the OCT4::EGFP fusion protein are derived from the transgenic mouse. In some cases, germline cells expressing the OCT4::EGFP fusion protein are derived from the transgenic mouse.

[0007] In some embodiments, also disclosed herein is a method of producing a transgenic mouse, the method comprising microinjecting a zygote with a bacterial artificial chromosome (BAC) construct, the construct comprising a reporter gene operably linked to the mouse OCT4 locus, and implanting the zygote into the reproductive tract of a surrogate mouse, thereby producing a transgenic mouse.

[0008] In some embodiments, also disclosed herein is a method for evaluating a product used for assisted reproductive technologies (ART), disease treatment, drug screening, or immune modulation, comprising the steps of: (a) obtaining a transgenic embryo comprising stable expression of a fusion protein comprising OCT4; (b) culturing the transgenic embryo; (c) evaluating expression of the fusion protein; and (d) determining the acceptability or failure of the product.

[0009] Further disclosed herein, in some embodiments, is a kit comprising a transgenic mouse described herein, an embryo described herein, a stem cell described herein, or a germline cell described herein, and optionally, instructions for use. [Brief description of the drawings]

[0010] [Figure 1-1] 1A-1B show the effect of suboptimal oil exposure on transgenic and control embryos described herein for 48 hours. FIG. 1A illustrates the study protocols. Method A refers to the study protocol using transgenic embryos described herein. Method B refers to the study protocol using control embryos. FIG. 1B shows a comparison of detected blastomeres between transgenic and control embryos described herein. [Figure 1-2] 1A-1B show the effect of suboptimal oil exposure on transgenic and control embryos described herein at 48 hours. FIG. 1A illustrates the study protocol. Method A refers to the study protocol using transgenic embryos described herein. Method B refers to the study protocol using control embryos. FIG. 1B shows a comparison of detected blastomeres between transgenic and control embryos described herein. [Figure 2-1] 2A-C show the effect of suboptimal conditions on cryopreserved transgenic and control embryos described herein. FIG. 2A illustrates the study design. Method A refers to the study protocol using transgenic embryos described herein. MEA refers to mouse embryo assay using control embryos. Comparison of detected blastomeres between transgenic and control embryos described herein is shown at 48 hours (FIG. 2B) and 96 hours (FIG. 2C). [Figure 2-2]2A-C show the effect of suboptimal conditions on cryopreserved transgenic and control embryos described herein. FIG. 2A illustrates the study design. Method A refers to the study protocol using transgenic embryos described herein. MEA refers to mouse embryo assay using control embryos. Comparison of detected blastomeres between transgenic and control embryos described herein is shown at 48 hours (FIG. 2B) and 96 hours (FIG. 2C). [Figure 2-3] 2A-C show the effect of suboptimal conditions on cryopreserved transgenic and control embryos described herein. FIG. 2A illustrates the study design. Method A refers to the study protocol using transgenic embryos described herein. MEA refers to mouse embryo assay using control embryos. Comparison of detected blastomeres between transgenic and control embryos described herein is shown at 48 hours (FIG. 2B) and 96 hours (FIG. 2C). [Diagram 3] Figure 3 shows the abnormal expression of OCT4-GFP in transgenic and control embryos cultured in expired ART medium A as described herein at 48 hours. Method A refers to the use of transgenic embryos as described herein. MEA refers to mouse embryo assay using control embryos. [Figure 4] Figure 4 shows the abnormal expression of OCT4-GFP in transgenic and control embryos cultured in expired ART medium A at 96 hours. Method A refers to the use of transgenic embryos described herein. MEA refers to mouse embryo assay using control embryos.

[0011] Detailed Description definition The embodiments according to the disclosure of the present invention will be described in more detail below. However, the aspects of the disclosure can be embodied in various forms, and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. The terms used in the description herein are merely for the purpose of describing specific embodiments, and are not intended to be limiting.

[0012] Unless otherwise specified, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted to have a meaning consistent with their meaning in the context of this application and the relevant art, and should not be interpreted in an idealized or overly formal sense unless expressly defined as such herein. Unless expressly defined below, such terms should be interpreted according to their ordinary meaning.

[0013] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0014] The practice of the techniques of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, within the skill of one of ordinary skill in the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0015] Unless otherwise indicated by the context, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composite comprises components A, B, and C, it is specifically intended that any or any combination of A, B, or C can be omitted or excluded, either alone or in any combination.

[0016] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations that are varied (+) or (-) in increments of 1.0 or 0.1, or alternatively with a variation of + / -15%, or alternatively with a variation of 10%, or alternatively with a variation of 5%, or alternatively with a variation of 2%, as appropriate. All numerical designations shall be understood, although not always expressly stated, to be preceded by the term "about". It shall also be understood, although not always expressly stated, that the reagents described herein are merely exemplary, and that equivalents of such are known in the art.

[0017] As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0018] The term "about" as used herein refers to a measurable value, such as an amount or concentration, and is meant to encompass a 20%, 10%, 5%, 1%, 0.5%, or even 0.1% variation of the specified amount.

[0019] The term "comprising," as used herein, is intended to mean that the compositions and methods include the recited elements, but do not exclude others. As used herein, the term "consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that have some essential importance to the combination. For example, a composition or method consisting essentially of the elements defined herein would not exclude other elements that do not materially affect the novel characteristics underlying the claimed invention. "Consisting of," as used herein, is intended to mean excluding more than trace amounts of other ingredients or substantial method steps recited. Embodiments defined by each of these transitional phrases are within the scope of this disclosure.

[0020] The terms "acceptable," "effective," or "sufficient," as used herein, refer to the intention that the selection of any components, ranges, dosage forms, etc. disclosed herein, is suitable for the purpose disclosed, said components, ranges, dosage forms, etc.

[0021] "And / or," as used herein, refers to and includes any and all possible combinations of one or more of the associated listed items, in addition to the exclusion of combinations when interpreted as either / or ("or").

[0022] The terms "nucleic acid sequence", "nucleic acid molecule", or "polynucleotide", as used herein, are used interchangeably and refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain, alternatively consist essentially of, or even consist of, purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0023] The term "enhancer" as used herein refers to a region of a DNA sequence that encodes a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence capable of providing both promoter and enhancer functions. For example, retroviral long terminal repeats contain both promoter and enhancer functions. An enhancer / promoter may be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in a genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in juxtaposition to a gene by means of genetic engineering (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. The term "promoter" as used herein refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, and that aids or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene.

[0024] "Under transcriptional control," as used herein, is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent upon it being operably linked to elements that contribute to or facilitate the initiation of transcription.

[0025] The term "polypeptide" as used herein refers to a chain of at least two covalently linked amino acids. A polypeptide may be encoded by a polynucleotide provided herein. A protein provided herein may be encoded by a nucleic acid sequence provided herein. A protein may comprise a polypeptide or an amino acid sequence provided herein. A "protein" as used herein refers to a chain of amino acid residues capable of providing a structure or enzymatic activity to a cell. A "coding sequence" as used herein refers to a nucleic acid sequence that encodes a protein.

[0026] As used herein, the term "encodes" when applied to a nucleic acid sequence refers to a polynucleotide that, in its natural state or when manipulated by methods well known to those of skill in the art, is said to "encode" a polypeptide when it can be transcribed and / or translated to produce mRNA for a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoded sequence can be deduced therefrom.

[0027] As used herein, the terms "equivalent" or "biological equivalent" or "similar" are used interchangeably when referring to a particular molecule, biological, or cellular material, and are intended to have minimal homology while still maintaining a desired structure or functionality. Non-limiting examples of equivalent polypeptides include polypeptides that have at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% identity to a given polypeptide or polypeptide sequence, or polypeptides encoded by polynucleotides or their complements that hybridize under high stringency conditions to polynucleotides encoding such polypeptide sequences. High stringency conditions are described herein and are incorporated by reference. Alternatively, those equivalents are polypeptides encoded by polynucleotides or their complements having at least 70%, or alternatively at least 75%, or alternatively 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or at least 97% sequence identity to a reference polynucleotide, e.g., to a wild-type polynucleotide.

[0028] The terms "operably linked" or "operably linked" as used herein intend that polynucleotides are positioned in a manner that allows them to function in the cell.

[0029] The term "gene," as used herein, also refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. "Gene product" or, alternatively, "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0030] The term "reporter gene" as used herein includes a gene that can be operably linked to a regulatory region of a survival marker and can be visualized or otherwise evaluated to determine its expression. In a preferred embodiment, the reporter gene is a fluorescent or luminescent protein. Fluorescent proteins can include, but are not limited to, blue / UV proteins such as TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire; cyan proteins such as ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFP1; green proteins such as EGFP, Emerald, Superfolder GFP, monomeric Azami Green, TagGFP2, mUKG, mWasabi, or Clover; yellow fluorescent proteins such as EYFP, Citrine, Venus, SYFP2, ZsYellow1, and TagYFP; orange proteins for use as reporter genes include monomeric Kusabira-Orange, mKO k, mKO2, mOrange, and mOrange2; red proteins, such as HcRed1, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, mApple, mRuby, and mRuby2; far-red proteins, such as, but not limited to, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP. In some embodiments, the fluorescent protein is selected from green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YPE), or cyan fluorescent protein (CFP). In some embodiments, the reporter gene may be, or may include, an epitope tag (e.g., HIS, FLAG, HA) recognized by an antibody.

[0031] The term "linker" as used herein refers to an amino acid or peptidomimetic sequence. In some embodiments, the linker has one or more properties, such as flexible conformation, inability to form ordered secondary structures, or flexible hydrophobic or charged features that can promote or interact with the activity of each domain. Amino acids typically found in flexible protein regions include, but are not limited to, Gly, Asn, and Ser. The length of the linker sequence can be altered without significantly affecting function or activity.

[0032] The term "fusion protein," as used herein, refers to a protein having at least two separately encoded domains joined together such that they are transcribed and translated as a single protein.

[0033] The term "mutation" as used herein refers to an alteration in the nucleotide sequence of the genome of an organism, a virus, or extrachromosomal DNA.

[0034] The term "stably expresses" or "stably express," as used herein, refers to the integration of a foreign gene into the genome.

[0035] The terms "C-terminus", "carboxyl terminus", "carboxy terminus", "C-terminal tail", "extreme C-terminus" or "COOH-terminus" as used herein refer to the end of an amino acid chain terminated by a free carboxyl group (-COOH). 2 "Terminal," "N-terminus," or "amine terminus," as used herein, refers to a free amine group (-NH 2 ) refers to the beginning of a chain of amino acids. When a protein is translated from messenger RNA, translation is from the N-terminus to the C-terminus.

[0036] The term "bacterial artificial chromosome construct" or "BAC construct" as used herein refers to a DNA construct used for transformation and cloning in bacteria.

[0037] The term "germline," as used herein, refers to the population of cells of a multicellular organism whose genetic material is passed on to progeny. In some embodiments, the germline is the cell that forms eggs, sperm, and zygotes.

[0038] The term "culturing," as used herein, refers to the in vitro propagation of cells or organisms on or in various types of media. It is understood that the descendants of cells propagated in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0039] The term "mammal" as used herein refers to any species classified within the class Mammalia.

[0040] The term "mouse" as used herein refers to Mus musculus.

[0041] The term "viable" as used herein refers to an animal or cell capable of surviving or living under particular environmental conditions.

[0042] The term "fertile" as used herein refers to the ability to produce offspring (offspring).

[0043] The term "offspring" or "progeny" as used herein refers to the younger generation born to an organism.

[0044] The term "reproductive organs" or "reproductive system" as used herein refers to the set of organs that contribute to and serve the reproductive process.

[0045] The term "surrogate," as used herein, refers to a female animal that is impregnated by embryo transfer or artificial insemination to provide offspring in place of another animal.

[0046] The term "transgenic" as used herein refers to a segment of DNA that is incorporated into a host genome or that can replicate in a host cell and express one or more cellular products. An exemplary transgene can provide a host cell or an animal developed therefrom with a novel phenotype compared to the corresponding non-transformed cell or animal. The term "transgenic animal" as used herein refers to a non-human animal, usually a mammal, that has a non-endogenous nucleic acid sequence present as an extrachromosomal element in at least some of its cells or stably integrated into its germline DNA. In some embodiments, the transgenic animal is a transgenic mouse.

[0047] Transgenesis is used to generate transgenic mammals, such as mice, with reporter genes linked to genes of interest. Methods in molecular genetics and genetic engineering are generally described in Molecular Cloning: A Laboratory Manual, (Sambrook et al.); Oligonucleotide Synthesis (MJ Gait, ed.); Animal Cell Culture (RI Freshney, ed.); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3.sup.rd Edition (FM Ausubel et al., eds.); and the latest edition of Recombinant DNA Methodology (R. Wu ed., Academic Press). Thus, transgenic technology is well established. See, e.g., Transgenic Mouse: Methods and Protocols (M. Hofker and J. Deursen, Eds.) in Methods in Molecular Biology (Vol. 209), the contents of which are incorporated herein by reference in their entirety.

[0048] The term "microinjection," as used herein, refers to the use of a glass micropipette to inject substances at the microscopic level.

[0049] As used herein, the term "assisted reproductive technology" or "ART" as used herein includes any procedure for conception in which both female gametes (eggs or oocytes) and male gametes (sperm) are manipulated. In vitro fertilization (IVF) is one of a number of assisted reproductive techniques used to assist infertile couples in having children. IVF refers to a procedure in which eggs are removed from a woman's ovaries and fertilized with sperm in an experimental procedure. The fertilized eggs (embryos) may be frozen for future use or transferred to the uterus.

[0050] "Morula" as used herein refers to an early stage embryo containing about 16 cells in the shape of a solid sphere contained within a zona pellucida. Morula is also referred to as a blastomere.

[0051] "Blastocyst," as used herein, refers to a structure in early embryonic development consisting of a sphere of cells with a surrounding wall (trophectoderm or TE) that will form the placenta, a fluid-filled cavity (blastocyst) that will form the amniotic cavity, and an inner cluster of cells called the inner cell mass (ICM) from which the fetus develops.

[0052] Octamer-binding transcription factor 4 (Oct-4 or OCT4; also referred to as POU domain, class 5, transcription factor 1 (POU5F1)), as used herein, is a protein involved in the self-renewal of undifferentiated embryonic stem cells. OCT4 contains three domains: the N-terminal domain, the POU domain, and the C-terminal domain. Both the N-terminal and C-terminal domains are involved in transactivation, but the activity of the C-terminal domain is cell type specific and regulated via phosphorylation. The POU-domain serves as an interaction site for binding by cell type-specific regulatory factors.

[0053] The Mouse Embryo Assay (MEA) is a functional and toxicological bioassay utilized to detect toxic and suboptimal compounds. The MEA remains the gold standard for testing the suitability of culture media and environments without involving human material. The basic techniques and protocols employed to perform the MEA are described in In Vitro Fertilization and Embryo Transfer: A Manual of Basic Techniques (Don P. Wolf, Editor), 1988, pages 57-75; and Mouse Embryo Assay for Assisted Reproduction Technology Devices: Guidance for Industry and Food and Drug Administration Staff, published by the U.S. Food and Drug Administration, the contents of which are incorporated herein by reference in their entirety. Briefly, the assay involves superovulation of female mice with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Mice are placed with males at the time of hCG injection and killed 24 hours after hCG to obtain 1-cell embryos or 36 hours after injection to obtain 2-cell embryos. 1-cell embryos are selected for use if they have two visible polar bodies; 2-cell embryos are selected for use if they appear morphologically normal. To examine whether the test article may present any toxicity to mouse embryos, the embryos are cultured under normal culture conditions (e.g., 37°C and 5% CO). 2 Embryos can be incubated in the test article under 500 ng / mL for about 96 hours if a one-cell line is used, or 72 hours if a two-cell line. Alternatively, culture may be extended for 5, 6, or longer days. Once embryo culture is complete, the embryos can be evaluated for development (e.g., blastocyst development). Acceptance may include when 80% or more of the embryos develop into expanded blastocysts.

[0054] Transgenic mice In certain embodiments, disclosed herein is a transgenic mouse that comprises, consists essentially of, or consists of stable expression of a fusion protein that comprises octamer-binding transcription factor 4 (OCT4). In some cases, the fusion protein is under transcriptional control. In some cases, the gene expression of the fusion protein is stably inherited through germline DNA.

[0055] In some embodiments, the OCT4 protein is mouse OCT4. The OCT4 protein may comprise full-length OCT4, or a fragment thereof, e.g., a functional fragment thereof. The term "functional fragment" as used herein refers to an OCT4 fragment capable of inducing an equivalent function to wild-type OCT4, such as transactivation, self-renewal of undifferentiated embryonic stem cells, and / or pluripotency of embryonic cells. In some cases, the OCT4 protein comprises a deletion (e.g., deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more residues) at the N-terminus, C-terminus, and / or internal region within the protein. In some cases, the OCT4 protein comprises a deletion of a domain, e.g., deletion of the N-terminus domain, the C-terminus domain, and / or the POU domain. In some cases, the OCT4 protein comprises a wild-type OCT4 protein. In other cases, the OCT4 protein contains one or more mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations.

[0056] The OCT4 protein may comprise at least or about 70% sequence identity or similarity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 80% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 90% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 95% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 96% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 97% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 98% sequence identity to SEQ ID NO: 1. In some cases, the OCT4 protein comprises at least or about 99% sequence identity to SEQ ID NO: 1. In some cases, the OCT4 protein comprises the sequence set forth in SEQ ID NO: 1. In some cases, the OCT4 protein consists of SEQ ID NO: 1.

[0057] In some embodiments, the fusion protein is a fluorescently tagged OCT4 protein. In some cases, the fluorescent tag is a fluorescent protein, including green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP). In some cases, the fluorescent protein is GFP or enhanced green fluorescent protein (eGFP). In some cases, the fluorescent protein is a wild-type protein, such as wild-type GFP or eGFP. In other cases, the fluorescent protein comprises one or more mutations, for example, comprises one or more mutations in GFP or eGFP.

[0058] In some embodiments, the fluorescent protein is GFP (e.g., eGFP). In some cases, the GFP (e.g., eGFP) is full-length GFP. In other examples, the GFP (e.g., eGFP) is a fragment thereof, e.g., a functional fragment thereof. The term "functional fragment" as used herein refers to a GFP fragment capable of producing fluorescence. In some cases, the GFP (e.g., eGFP) comprises deletions (e.g., deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more residues) at the N-terminus, C-terminus, and / or internal regions within the protein. In some cases, the GFP (e.g., eGFP) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some cases, the GFP (e.g., eGFP) comprises an A206K mutation.

[0059] In some cases, the fluorescent protein is a GFP that comprises at least or about 70% sequence identity or similarity with SEQ ID NO:2. In some cases, the GFP comprises at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity with SEQ ID NO:2. In some cases, the GFP comprises at least or about 80% sequence identity with SEQ ID NO:2. In some cases, the GFP comprises at least or about 90% sequence identity with SEQ ID NO:2. In some cases, the GFP comprises at least or about 95% sequence identity with SEQ ID NO:2. In some cases, the GFP comprises at least or about 96% sequence identity with SEQ ID NO:2. In some cases, the GFP comprises at least or about 97% sequence identity with SEQ ID NO:2. In some cases, GFP comprises at least or about 98% sequence identity to SEQ ID NO: 2. In some cases, GFP comprises at least or about 99% sequence identity to SEQ ID NO: 2. In some cases, GFP comprises the sequence set forth in SEQ ID NO: 2. In some cases, GFP consists of SEQ ID NO: 2.

[0060] The fluorescent protein (e.g., GFP or eGFP) may be operably linked to the N-terminus, C-terminus, or internal site of the OCT4 protein. In some cases, the fluorescent protein (e.g., GFP or eGFP) is operably linked to the C-terminus of the OCT4 protein.

[0061] In some embodiments, the germline is selected from, but not limited to, sperm, oocytes, stem cells, or zygotes. In some cases, the germline is selected from sperm. In some cases, the germline is selected from oocytes. In some cases, the germline is selected from stem cells. In some cases, the germline is selected from a zygote.

[0062] In some cases, the transgenic mouse is a viable and fertile mouse. In some cases, the transgenic mouse is a viable male capable of producing offspring that contain the fusion protein stably incorporated into the offspring. In other examples, the transgenic mouse is a viable female capable of producing offspring that contain the fusion protein stably incorporated into the offspring.

[0063] In some cases, gene expression of the fusion protein in the zygote begins at the developing two-cell, three-cell, or four-cell stage cells.

[0064] In certain embodiments, disclosed herein is a method for producing the transgenic mouse described above. In some embodiments, the method comprises microinjecting a zygote with a construct that comprises, alternatively consists essentially of, or even consists of a reporter gene operably linked to mouse OCT4 locus, and implanting the zygote into the reproductive organs of a surrogate mouse, thereby producing a transgenic mouse. In some cases, the construct is a bacterial artificial chromosome (BAC) construct, and the construct comprises, alternatively consists essentially of, or even consists of a reporter gene operably linked to mouse OCT4 locus. In some cases, the transgenic mouse stably expresses the reporter gene.

[0065] In some embodiments, the reporter locus is stably inherited through the germline DNA of the transgenic mouse. The germline can be selected from sperm, oocytes, stem cells, or zygotes.

[0066] In some embodiments, the reporter gene encodes a fluorescent protein. In some cases, the fluorescent protein is selected from, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP). In one embodiment, the GFP is enhanced green fluorescent protein (eGFP). In one embodiment, the eGFP comprises, alternatively essentially consists of, or even consists of A206K mutation.

[0067] In some embodiments, the reporter gene comprises a nucleic acid sequence encoding a fluorescent protein that comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 80% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 85% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 90% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 95% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 96% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 97% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 98% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises at least or about 99% sequence identity to SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein that comprises SEQ ID NO:2. In some cases, the nucleic acid sequence encodes a fluorescent protein consisting of SEQ ID NO:2.

[0068] In some embodiments, the reporter gene is operably linked to the coding sequence. In one aspect, the coding sequence encodes an OCT4 protein. In some cases, the OCT4 protein comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 80% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 90% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 95% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 96% sequence identity with SEQ ID NO:1. In some cases, the OCT4 protein comprises at least or about 97% sequence identity to SEQ ID NO: 1. In some cases, the OCT4 protein comprises at least or about 98% sequence identity to SEQ ID NO: 1. In some cases, the OCT4 protein comprises at least or about 99% sequence identity to SEQ ID NO: 1. In some cases, the OCT4 protein comprises the sequence set forth in SEQ ID NO: 1. In some cases, the OCT4 protein consists of SEQ ID NO: 1.

[0069] In some embodiments, the reporter gene and the gene coding sequence (e.g., OCT4) are separated by a linker. In one embodiment, the linker encodes an amino acid sequence that includes multiple Ala, Gly, or a combination thereof. In one embodiment, the linker is 4The present invention relates to a method for producing a reporter gene comprising the steps of: encoding an amino acid sequence comprising a linker Ser, wherein n is an integer selected from 1-10; optionally an integer selected from 1-6, 1-4, and 1-3; and further optionally an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment, the linker encodes an amino acid sequence comprising SGGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the reporter gene is operably linked to the N-terminus, C-terminus, or internal region of the coding sequence (e.g., OCT4). In one embodiment, the linker connects the reporter gene to the C-terminus of the coding sequence (e.g., OCT4).

[0070] In some embodiments, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 70% sequence identity or similarity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 80% sequence identity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 90% sequence identity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 95% sequence identity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 96% sequence identity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 97% sequence identity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 98% sequence identity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises at least or about 99% sequence identity with SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein comprises the sequence set forth in SEQ ID NO:4. In some cases, the polypeptide comprising the fluorescent protein and OCT4 protein consists of SEQ ID NO:4.

[0071] In some embodiments, the construct encodes an OCT4::EGFP fusion protein. In some cases, the construct comprises a nucleic acid sequence that comprises at least or about 70% sequence identity or similarity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or similarity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 80% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 85% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 90% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 95% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 96% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 97% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 98% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises at least or about 99% sequence identity to SEQ ID NO:5. In some cases, the nucleic acid sequence comprises the sequence set forth in SEQ ID NO:5. In some cases, the nucleic acid sequence consists of SEQ ID NO:5.

[0072] In some cases, the construct mediates expression of an OCT4::EGFP fusion protein. In some cases, the OCT4::EGFP fusion protein is stably integrated into the zygote.

[0073] In some cases, the OCT4 locus in the construct contains a deletion of the proximal enhancer element.

[0074] In some embodiments, disclosed herein is an embryo expressing an OCT4::EGFP fusion protein, wherein an oocyte is fertilized with sperm containing the OCT4::EGFP fusion protein, and the sperm is derived from the transgenic mouse described above.

[0075] In some embodiments, disclosed herein are stem cells expressing an OCT4::EGFP fusion protein derived from the transgenic mice described above.

[0076] In some embodiments, disclosed herein are germline cells expressing an OCT4::EGFP fusion protein derived from the transgenic mice described above.

[0077] Methods for product evaluation In certain embodiments, disclosed herein is a method for evaluating a product used for assisted reproductive technology (ART), disease treatment, drug screening, or immune modulation.In some cases, the method includes the steps of (a) obtaining a transgenic embryo comprising stable expression of a fusion protein comprising OCT4; (b) culturing the transgenic embryo; (c) evaluating the expression of the fusion protein; and (d) determining the acceptability or failure of the product.

[0078] In some embodiments, the fusion protein is a fluorescent protein fused to OCT4 protein.In some cases, the fluorescent protein is selected from green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP).In some cases, the fluorescent protein is selected from GFP or enhanced green fluorescent protein (eGFP).In some cases, eGFP comprises a mutation, for example, comprises an A206K mutation.

[0079] In some embodiments, the evaluating step includes determining the temporal and / or spatial expression pattern of the fusion protein. The evaluating step may include visualizing the nuclear and / or cytoplasmic localization of the fusion protein. The nuclear localization may include shuttling of the fusion protein to the nucleus as well as binding of DNA by the fusion protein in the nucleus. The evaluating step may further include comparing the temporal and / or spatial expression pattern of the fusion protein with a control to determine whether an abnormality occurs in embryo development. Control as used herein refers to the temporal and / or spatial expression pattern of the fusion protein from a comparable embryo in which the embryo has undergone normal development.

[0080] In some cases, the evaluating step is performed at the 4-cell or 8-cell stage. In some cases, the fusion protein is predominantly localized in the nucleus at the 4-cell stage. The term "predominantly" as used herein refers to at least or about 50%, 60%, 70%, 80%, 90%, 95% or more of the fusion protein being localized in the nucleus. In some cases, at least or about 50% of the fusion protein is localized in the nucleus. In some cases, at least or about 60% of the fusion protein is localized in the nucleus. In some cases, at least or about 70% of the fusion protein is localized in the nucleus. In some cases, at least or about 80% of the fusion protein is localized in the nucleus. In some cases, at least or about 90% of the fusion protein is localized in the nucleus. In some cases, at least or about 95% of the fusion protein is localized in the nucleus.

[0081] In some cases, the evaluating step includes determining the location of expression of the fusion protein at the 4-cell stage or the 8-cell stage. In some cases, the fusion protein is predominantly expressed in the nucleus at the 4-cell stage (e.g., at least or about 50%, 60%, 70%, 80%, 90%, 95% or more of the fusion protein expressed in the nucleus). In some cases, at least or about 50% of the fusion protein is expressed in the nucleus. In some cases, at least or about 60% of the fusion protein is expressed in the nucleus. In some cases, at least or about 70% of the fusion protein is expressed in the nucleus. In some cases, at least or about 80% of the fusion protein is expressed in the nucleus. In some cases, at least or about 90% of the fusion protein is expressed in the nucleus. In some cases, at least or about 95% of the fusion protein is expressed in the nucleus.

[0082] In some cases, the evaluating step is performed at the 8-cell stage. In some cases, at least or about 80%, 90%, 95%, 99% or more of the fusion protein is localized in the nucleus. In some cases, at least or about 80% of the fusion protein is localized in the nucleus. In some cases, at least or about 90% of the fusion protein is localized in the nucleus. In some cases, at least or about 95% of the fusion protein is localized in the nucleus. In some cases, about 100% of the fusion protein is localized in the nucleus.

[0083] In some cases, the evaluating step includes determining the location of expression of the fusion protein at the 8-cell stage. In some cases, at least or about 80%, 90%, 95%, 99%, or more of the fusion protein is expressed in the nucleus. In some cases, at least or about 80% of the fusion protein is expressed in the nucleus. In some cases, at least or about 90% of the fusion protein is expressed in the nucleus. In some cases, at least or about 95% of the fusion protein is expressed in the nucleus. In some cases, about 100% of the fusion protein is expressed in the nucleus.

[0084] In some cases, the evaluating step is performed at the morula stage. In some cases, at least or about 80%, 90%, 95% or more of the fusion protein is localized in the nucleus. In some cases, at least or about 80% or more of the fusion protein is localized in the nucleus. In some cases, at least or about 90% or more of the fusion protein is localized in the nucleus. In some cases, at least or about 95% or more of the fusion protein is localized in the nucleus. In some cases, about 100% of the fusion protein is localized in the nucleus.

[0085] In some cases, the evaluating step includes determining the location of expression of the fusion protein at the morula stage. In some cases, at least or about 80%, 90%, 95% or more of the fusion protein is expressed in the nucleus. In some cases, at least or about 80% or more of the fusion protein is expressed in the nucleus. In some cases, at least or about 90% or more of the fusion protein is expressed in the nucleus. In some cases, at least or about 95% or more of the fusion protein is expressed in the nucleus. In some cases, about 100% of the fusion protein is expressed in the nucleus.

[0086] In some cases, the evaluating step is performed at the blastocyst stage. In some cases, at least or about 60%, 70%, 80%, 90%, 95% or more of the fusion protein is localized in the inner cell mass (ICM). In some cases, at least or about 70% or more of the fusion protein is localized in the ICM. In some cases, at least or about 80% or more of the fusion protein is localized in the ICM. In some cases, at least or about 90% or more of the fusion protein is localized in the ICM. In some cases, at least or about 95% or more of the fusion protein is localized in the ICM. In some cases, about 100% of the fusion protein is localized in the ICM. In some cases, the fusion protein is not localized in the trophoblast.

[0087] In some cases, the evaluating step includes determining the location of expression of the fusion protein at the blastocyst stage. In some cases, at least or about 60%, 70%, 80%, 90%, 95% or more of the fusion protein is expressed in the inner cell mass (ICM). In some cases, at least or about 70% or more of the fusion protein is expressed in the ICM. In some cases, at least or about 80% or more of the fusion protein is expressed in the ICM. In some cases, at least or about 90% or more of the fusion protein is expressed in the ICM. In some cases, at least or about 95% or more of the fusion protein is expressed in the ICM. In some cases, about 100% of the fusion protein is expressed in the ICM. In some cases, the fusion protein is not expressed in the trophoblast.

[0088] In some embodiments, the fusion protein is detectable at about 24 hours to about 96 hours, about 24 hours to about 72 hours, about 24 hours to about 48 hours, about 24 hours to about 36 hours, about 36 hours to about 96 hours, about 36 hours to about 72 hours, about 36 hours to about 48 hours, about 48 hours to about 72 hours, or about 48 hours to about 96 hours of culture. In some cases, the fusion protein is detectable at about 36 hours to about 96 hours of culture. In some cases, the fusion protein is detectable at about 36 hours to about 72 hours of culture. In some cases, the fusion protein is detectable at about 36 hours to about 48 hours of culture. In some cases, the fusion protein is detectable at about 48 hours to about 96 hours of culture. In some cases, the fusion protein is detectable at about 48 hours to about 72 hours of culture. In some cases, the fusion protein is detectable via visual inspection, for example, based on the fluorescence of the fluorescent protein. In other examples, the fusion protein is detected via nucleic acid expression analysis. In additional examples, the fusion protein is detected via protein expression analysis.

[0089] In some cases, the fusion protein is detectable at about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours of culture. In some cases, the fusion protein is detectable at about 36 hours of culture. In some cases, the fusion protein is detectable at about 48 hours of culture. In some cases, the fusion protein is detectable at about 72 hours of culture. In some cases, the fusion protein is detectable at about 96 hours of culture. In some cases, the fusion protein is detected via visual inspection, for example, based on the fluorescence of a fluorescent protein. In other examples, the fusion protein is detected via nucleic acid expression analysis. In additional examples, the fusion protein is detected via protein expression analysis.

[0090] In some embodiments, the fusion protein is detectable at 2-cell stage, 3-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, morula stage, or blastocyst stage. In some embodiments, the fusion protein is detectable at 2-cell stage, 3-cell stage, 4-cell stage, or 8-cell stage cell development. In some cases, the fusion protein is detectable at 4-cell stage cell development. In some cases, the fusion protein is detectable at 8-cell stage cell development. In some cases, the fusion protein is detectable at 16-cell stage cell development. In some cases, the fusion protein is detectable at morula stage cell development. In some cases, the fusion protein is detectable at blastocyst stage cell development. In some cases, the fusion protein is detected via visual inspection, for example, based on the fluorescence of a fluorescent protein. In other examples, the fusion protein is detected via nucleic acid expression analysis. In additional examples, the fusion protein is detected via protein expression analysis.

[0091] In some cases, the evaluating step is performed once a day, twice a day, three times a day, every other day, or on each successive day during the culture process. In some cases, one or more evaluating steps are performed from about 24 hours to about 96 hours, about 24 hours to about 72 hours, about 24 hours to about 48 hours, about 24 hours to about 36 hours, about 36 hours to about 96 hours, about 36 hours to about 72 hours, about 36 hours to about 48 hours, about 48 hours to about 72 hours, or about 48 hours to about 96 hours from the start of the culture process.

[0092] In some embodiments, the evaluating step may include, for example, one or more of: a) capturing at least one image of the transgenic embryo at a particular developmental stage; b) determining the location of the fusion protein based on the image; and c) comparing the location of the fusion protein to a control, which may be the location of the fusion protein in a comparable transgenic embryo at a particular developmental stage, the comparable transgenic embryo having undergone normal embryonic development.

[0093] In some embodiments, the evaluating step further comprises determining the expression level of the fusion protein along with a control. In some cases, the expression level is determined by determining nucleic acid expression, by determining protein expression, by measuring light emission and / or intensity, visually or using a device therefor.

[0094] In some cases, for example, if there is nuclear localization or expression of the fusion protein at the 4-cell, 8-cell, or morula stage, the product is accepted. In some cases, if there is localization or expression in the ICM during the blastocyst stage, the product is accepted.

[0095] In some cases, if there is less than 40%, 30%, 20%, 10%, 5%, or 1% nuclear localization or expression of the fusion protein at the 4-cell or 8-cell stage, the product is not acceptable. In some cases, if there is no nuclear localization or expression of the fusion protein at the 8-cell stage, the product is not acceptable.

[0096] In some cases, if there is less than 40%, 30%, 20%, 10%, 5%, or 1% nuclear localization or expression of the fusion protein at the morula stage, the product is unacceptable. In some cases, if there is no nuclear localization or expression of the fusion protein at the morula stage, the product is unacceptable.

[0097] In some cases, if there is less than 40%, 30%, 20%, 10%, 5%, or 1% localization or expression of the fusion protein in the ICM at the blastocyst stage, the product is not acceptable. In some cases, if there is no localization or expression of the fusion protein in the ICM at the blastocyst stage, the product is not acceptable. In some cases, if there is localization or expression of the fusion protein in the trophoblast at the blastocyst stage, the product is not acceptable.

[0098] In some embodiments, the product is for use with assisted reproductive technology (ART).Products include consumables, including but not limited to media, media supplements, plasticware, tubing, pipettes, pipette tips, etc., or any material that comes into contact with eggs or embryos.Plastic and glassware can include assisted reproduction needles, laboratory gloves, assisted reproduction catheters, and assisted reproduction microtools, such as pipettes, or other devices used in laboratories to expose, micromanipulate, hold, or move embryos.IVF consumables also include assisted reproduction laboratory equipment, including but not limited to syringes, IVF tissue culture dishes, IVF tissue culture plates, pipette tips, dishes, plates, and other containers that come into physical contact with gametes, embryos, or tissue culture medium. IVF consumables, as used herein, can include water purification systems intended to produce high quality sterile pyrogen-free water for reconstituting media used in aspirating, incubating, transferring or storing embryos for IVF or other assisted reproductive procedures, as well as for use as a final rinse for laboratory equipment or other assisted reproductive devices expected to come into contact with embryos. In some cases, the products include needles, catheters, microtools, laboratory equipment, syringes, tissue culture dishes, tissue culture plates, pipette tips, dishes, plates, water, water purification systems, media, media supplements, or other devices or reagents that come into physical contact with embryos.

[0099] In some embodiments, the method for evaluating products used for assisted reproductive technology (ART) can reduce the variability of morphology-based embryo grading. In some cases, the method allows visualization of the nuclear localization of fusion proteins, optionally after 48 hours of embryo culture. In some cases, the method can reduce false positives compared to comparable assays, such as mouse embryo assays (MEA).

[0100] In some embodiments, the product is a protein or gene associated with a disease. The product also includes a transgenic mouse containing the protein or gene for use as a mouse model. The disease may be cancer. In some cases, the cancer is a solid tumor. In other cases, the cancer is a hematological tumor. The protein or gene may be associated with cancer, optionally associated with a solid tumor or a hematological tumor. The protein or gene may be a tumor-associated antigen. Exemplary tumor-associated antigens include, but are not limited to, CD19; CD20; CD22 (Siglec 2); CD37; CD123; CD22; CD30; CD171; CS-1; epidermal growth factor receptor (EGFR); epidermal growth factor receptor variant III (EGFRvIII); human epidermal growth factor receptor (HER1); ganglioside G2 (GD2); TNF receptor family member B cell maturation (BCMA); prostate specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); or tumor-associated glycoprotein 72 (TAG72). The protein or gene may also be a protein or gene that is overexpressed or suppressed in a cancer subject compared to the expression of the protein or gene in a normal subject.

[0101] In some cases, the product is a protein or gene associated with an autoimmune disease, and / or a transgenic mouse containing the protein or gene for use as a mouse model. The protein or gene may be overexpressed or suppressed in a subject suffering from an autoimmune disease compared to the expression of the protein or gene in a normal subject.

[0102] In some embodiments, the product is a protein or gene related to embryo development.The protein or gene may be related to regulating protein-protein interaction or gene expression, metabolic process, cell morphogenesis, cell division, cell proliferation, DNA replication, cell differentiation, or DNA repair and transcription.The protein or gene may be related to cell communication, apoptosis, immune response, housekeeping, or tissue-specific function. Exemplary proteins or genes include, but are not limited to, pluripotent stem cell (PS) specific markers, such as the family of Sox genes (e.g., Sox1, Sox2, Sox3, Sox15, and Sox18); the family of Klf genes, such as Klf4 and Klf5; or the family of Nanog genes, such as NANOG; markers associated with the TGF-beta superfamily and their respective receptors; markers associated with the cryptic protein family (e.g., Cripto-1); markers associated with the integrin family (e.g., integrin alpha 6 (CD49f) and integrin beta 1 (CD29)); the podocalyxin family (PODX-1), the FGF family (e.g., FGF-β family), the markers associated with the integrin family (e.g., integrin alpha 6 (CD49f) and integrin beta 1 (CD29)); Examples of markers that may be mentioned include markers related to the FGF-1 family (e.g., FGF4 and FGF-5), the forkhead box transcription factor family (e.g., FoxD3), the T-box family of transcription factors (e.g., TBX3 and TBX5), the family of developmental pluripotency-associated molecules (e.g., Dppa2, Dppa3 / Stella, Dppa4 and Dppa5 / ESG1), the LRR family (e.g., 5T4), the cadherin family (e.g., E-cadherin), the connexin family of transmembrane proteins (e.g., connexin-43 and connexin-45), the F-box family of the "other" category (e.g., FBOXO15), the family of chemokines / chemokine receptors (e.g., CCR4 and CXCR4), or ATP-binding cassette transporters (e.g., ABCG2).

[0103] In some embodiments, one or more embryonic stem cells can also be obtained from transgenic embryo. One or more embryonic stem cells can be cultured to generate a plurality of embryonic stem cells. The plurality of embryonic stem cells can then be cultured with a drug. The expression of fusion protein can also be evaluated to determine the acceptability or failure of the drug. In some cases, the drug is for use in the treatment of disease, optionally cancer or autoimmune disease. In some cases, the drug is for use in modulating immune response.

[0104] Qualitative analysis of embryo development can be achieved by visually analyzing developing embryos, for example, by evaluating color, light intensity or fluorescence using optical microscopy, which may include ultraviolet light to visualize fluorescent protein expression. Confocal microscopy can also be used to evaluate developing embryos. In some cases, embryo development is observed through an embryoscope (e.g., EmbryoScope® Time-lapse System, Unisense Fertilitech A / S), where pictures of developing embryos can be taken, for example, approximately every 5, 10, 20, 30 seconds or more every few minutes as required, and time-lapse videos can be created to track all stages of embryo development.

[0105] Kits and articles of manufacture In certain embodiments, the present disclosure provides kits for carrying out the methods of the disclosure, as well as instructions for carrying out the methods of the present disclosure, which kits comprise, alternatively consist essentially of, or even consist of one or more of the following: constructs for introducing the fusion proteins described above, modified eggs (e.g., oocytes and / or zygotes), transgenic embryos, and / or transgenic mice described above, and instructions for use.

[0106] The kit may include culture media and / or supplements for use with the methods of this disclosure. In some cases, culture media include, but are not limited to, reproductive media and supplements used in assisted reproduction procedures. Media may include liquid and powder versions of various substances that will be in direct physical contact with embryos for preparation, maintenance, transportation, or storage purposes (e.g., water, acid solutions used to treat gametes or embryos, rinse solutions, reagents, semen separation medium, or oils used to cover the medium). Supplements may include specific reagents, such as proteins, serum, antibiotics, etc., that are added to the medium to enhance specific properties of the medium. Where appropriate, these suggested kit components may be packaged in a manner that is conventional for use by those skilled in the art.

[0107] Partial sequence listing OCT4 protein sequence - SEQ ID NO:1

[0108] [ka]

[0109] GFP protein sequence - SEQ ID NO:2

[0110] [ka]

[0111] OCT4::EGFP fusion protein sequence - SEQ ID NO:4

[0112] [ka]

[0113] Construct sequence - SEQ ID NO:5

[0114] [ka]

[0115] [ka]

[0116] [ka]

[0117] [ka]

[0118] [ka]

[0119] [ka]

[0120] [ka]

[0121] [ka] EXAMPLES

[0122] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0123] Example 1. Production of OCT4-GFP transgenic mice material and method BAC cloning and microinjection A bacterial artificial chromosome (BAC) construct was used for expression of Oct4 fusion proteins. Monomeric EGFP was recombineered into the Oct4(Pou5f1) locus. EGFP was inserted at the C-terminus of OCT4. To minimize steric hindrance between the reporter protein and OCT4, a flexible amino acid linker coding sequence (S(GGGGS) 3 ;SEQ ID NO:3) was inserted between the gene coding sequence and the reporter gene.

[0124] B6SJLF1 (Jackson Laboratory, Bar Harbor, ME) female egg donors were used. Following sequential injections of PMSG (5U per animal, 3 days prior to harvest, at noon: Prospec, Rehovot, Israel, #HOR-272) and hCG hormone (5U per animal, 1 day prior to harvest, at noon, SIGMA, St. Louis MO, #CG5-1VL). Females were mated with B6SJLF1 males the day prior to harvest. B6SJLF2 embryos were harvested on E0.5 and injected into the pronucleus with BAC constructs for each transgene. Injected embryos were implanted into the reproductive tract of pseudopregnant surrogate mothers (ICR: Charles River, Wilmington, MA). Twenty days after implantation, the number of newborns was counted and toe biopsies were performed at 7–10 days of age to extract DNA for PCR genotyping.

[0125] Genotyping Two PCR methods for genotyping were utilized: conventional PCR and qPCR. For conventional PCR, the annealing temperature was 58° C. The following primers were used to detect the eGFP sequence:

[0126] eGFP (product size = 227bp)

[0127] TMF738 forward: 5'-ATCTTCTTCAAGGACGACGGCAAC-3' (SEQ ID NO: 6)

[0128] TMF739 reverse: 5'-TCCTCGATGTTGTGGCGGATCTTG-3' (SEQ ID NO: 7)

[0129] Internal control (mouse Fndc3a gene: product size = 400 bp)

[0130] TMF725 forward: 5'-GAGCTTCTGGTATTAGCGTTAGGT-3' (SEQ ID NO: 8)

[0131] TMF726 reverse: 5'-TCCACAATGACAAAGACATGAGGT-3' (SEQ ID NO:9).

[0132] Taqman qPCR protocols were used to set up the CFX-BioRAD qPCR. The genotype of the EGFP transgene was determined by comparing the δCt values ​​of EGFP against known homozygous (HO) and hemizygous (HEMI) controls and an endogenous reference (ApoB gene).

[0133] The following PCR conditions were used: 95°C for 3 min -> (95°C for 15 sec -> 60°C for 30 sec) x 40 cycles.

[0134] Table 1 illustrates the qPCR primers and probes used.

[0135] [Table 1]

[0136] G0 backcross To isolate the transgenic allele, PCR-positive G0 founders were backcrossed to B6SJLF1 animals. Oct4-GFP offspring were backcrossed to the G3 generation to stabilize the transgene copy number.

[0137] Retrieval of embryos for imaging Hemizygous (HEMI) males were crossed with B6J females, and HEMI females were crossed with B6J males or Tg(Pou5f1-EGFP)2Mnn / J (Jackson Laboratory, Cat. No. 004654:TgOG2) HO males. B6J and TgOG2 females were superovulated by subsequent hormone injections (PMSG: 3 days prior to mating, and 5U of hCG: 1 day prior to mating). Animals were housed together overnight (for 1-cell embryo recovery) or for 2 days (2-cell stage embryo recovery). Embryos were collected and incubated at 37°C, 5% CO in a PLANER BT-37 incubator (Origio, Malov, Denmark). 2 , 5% O 2 , 90%N 2 and cultured in KSOM droplets overlaid with equilibrated mineral oil.

[0138] Imaging A Nikon microscope was used. Magnification was set at 11.5x. To compare signal intensity between litters or individual embryos, fluorescent imaging parameters were fixed at the same gain / exposure time. Brightfield images were taken with autoexposure settings.

[0139] Sperm cryopreservation Cryopreservation of semen was performed using established protocols as exemplified in Nakagata, N. (2011) Cryopreservation of Mouse Spermatozoa and In Vitro Fertilization. In: Hofker M., van Deursen J. (eds) Transgenic Mouse Methods and Protocols. Methods in Molecular Biology (Methods and Protocols), vol 693. Humana Press.

[0140] result Microinjections were performed using B6SJLF2 fertilized oocytes as the donor strain. 142 embryos were injected and 36 pups were born, confirming that the 7G0 animals carried the transgene.

[0141] To isolate the transgenic alleles, seven positive G0 animals were backcrossed with wild-type B6SJLF1 animals, respectively. Five of the seven founders inherited the transgene array through the germline (the subsequent lines or offspring from the five founders were named line A, line B, line C, line D, or line E, respectively). First, genotyping was performed by conventional PCR to detect the mEGFP insertion in the mouse genome. After differences in mEGFP expression intensity were observed in each line, a qPCR-dCT assay was deployed to measure the relative copy number of mEGFP in each line.

[0142] During breeding to generate independent lines, unusual variation in transgene copy number in the B line was observed between generations. Even in the G2 generation, variation in mEGFP copy number was observed within litters. The cause of transgene copy number variation remains unclear. However, after backcrossing of the transgene to B6SJL F1 wild-type mice in each line for at least two generations, the copy number variation disappeared. The variation may have occurred by intrachromosomal recombination involving the transgene array.

[0143] Relative mEGFP copy numbers were determined by normalizing the mEGFP signal to an internal control (diploid copies of the ApoB gene).

[0144] To determine whether HO mice were viable and fertile, G3 HEMI males were crossed with G3 HEMI females to further attempt to increase the OCT4-mEGFP signal. Genotypes were determined by qPCR-dCT methods: HO genotypes were determined by double doses of the GFP transgene compared to HEMI controls of each line. HO animals were confirmed as viable in lines A, B, C, and E. Chi-square analysis indicates that the genotypes of offspring from HEMI crosses in line C follow the expected Mendelian ratio. Results confirmed that line A HO embryos had increased viability compared to HEMI and WT, line B HO embryos had decreased viability, and line A and C HO were fertile. Line B HOs could mate and produce embryos, but line B HO females did not produce any pups when crossed with line B HO or HEMI males.

[0145] HEMI or HO males were crossed with superovulated B6J females (sire line). Embryos were collected and observed for mEGFP signal by conventional fluorescent stereomicroscope or confocal fluorescent microscope. Embryos were collected from five lines. GFP expression was examined under Nikon stereomicroscope. Embryos were cultured from 1-cell stage to blastocyst stage and observed daily. Expression was observed from 8-cell stage (96 hours) to blastocyst stage (120 hours). This was similar to GFP expression in OG2. Expression levels in each line were proportional to their mEGFP copy number. B line had the highest GFP expression level and D line had the lowest expression level. mEGFP expression was observed in a spotted pattern in each cell. This pattern was clearly different from OG2GFP. This was due to the IS construct having mEGFP fused to OCT4, rather than mEGFP simply produced from the Oct4 promoter as in the case of OG2 line.

[0146] Example 2 Development of a quantitative bioassay for assisted reproductive technology Pou5f1-GFP transgenic mouse lines expressing GFP-tagged POU5F1 were generated to exploit the nuclear localization of POU5F1 to detect deleterious culture conditions and epigenetic defects during the preimplantation period. Pou5f1-GFP expression was also used to visualize nuclei of blastomeres for cell counting in live cells. Pou5f1-GFP embryos were cultured for 96 h overlaid with optimal or suboptimal oil and POU5F1-GFP expression was observed at different stages of mouse embryo development (from 2PN to expanded / hatching blastocyst). (Experiments, n>3).

[0147] Pou5f1-GFP 1-cell embryos (fresh or frozen) were cultured to blastocyst stage in Continuous Single Culture Medium-Complete (CSCM-C, FUJIFILM Irvine Scientific) layered on top with control or suboptimal oil (oil containing 5, 7.5, or 10% impurities) in test conditions uninterrupted for up to 96 h and observed daily. B6 1-cell embryos, typically used in standard mouse embryo assays (MEA), were also cultured in parallel. These embryos were evaluated at 48 h (% of 8 cells or more) and 96 h (% of blastocysts).

[0148] Transgenic mice expressing Pou5f1-GFP were viable and fertile, confirming successful germline inheritance and temporally and spatially regulated gene expression. Zygote Pou5f1-GFP gene expression began around the 4-cell stage and peaked after 72 h of culture. Nuclear localization of POU5F1-GFP in mouse embryos allowed visualization of blastomere nuclei and cell counting in live cells as soon as GFP expression was detected around the 4-cell stage. Pou5f1-GFP embryos cultured with suboptimal oil overlay showed significant developmental delay (at 48 and 96 h compared to the control oil group). Mosaic patterned expression of POU5F1-GFP was observed in some embryos cultured with suboptimal oil overlay. Pou5f1-GFP embryo culture detected 5, 7.5, and 10% suboptimal conditions with statistical significance, while standard MEA (≥80% pass criteria) passed 5% suboptimal conditions at a rate of 28.3%. See Figures 1A-1B and 2A-2C. There was no difference in performance between fresh and frozen Pou5f1-GFP embryos. Pou5f1-GFP embryos cultured in deleterious culture conditions resulted in reduced subjectivity in grading embryos causing deleterious epigenetic effects. See Figures 3 and 4.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0150] The technology of the present invention described herein by way of example can be suitably carried out in the absence of any element or elements, or one or more limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc., are to be understood expansively and without limitation. In addition, the terms and expressions employed herein are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents or portions of the features shown or described, but it is recognized that various modifications are possible within the scope of the technology of the present invention as claimed.

[0151] Accordingly, it will be understood that the materials, methods, and examples provided herein are representative of preferred embodiments, are illustrative, and are not intended as limitations on the scope of the inventive technology.

[0152] The present technology has been described broadly and generally herein. Each of the narrower species and subgroups falling within the scope of the generic disclosure also constitutes part of the present technology. This includes the generic description of the present technology, but excludes any subject matter from the generic concept by a provisory or negative limitation, regardless of whether the carved out material is specifically recited herein.

[0153] In addition, where features or aspects of the present technology are described in terms of Markush groups, those of skill in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0154] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0155] Other aspects are defined in the following claims.

Claims

**Claim 1** A transgenic mouse comprising stable expression of a fusion protein comprising octamer-binding transcription factor 4 (OCT4) under transcriptional control, wherein gene expression of the fusion protein is stably inherited via germline DNA. **Claim 2** The transgenic mouse according to claim 1, wherein the fusion protein is OCT4 with a fluorescent tag. **Claim 3** The fluorescent tag is a fluorescent protein selected from green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP), Optionally, the fluorescent protein is GFP or enhanced green fluorescent protein (eGFP) optionally comprising an A206K mutation, according to claim 1 or 2. **Claim 4** The transgenic mouse according to claim 3, wherein the fluorescent protein comprises at least 70% sequence identity or similarity with SEQ ID NO: 2, or a fragment thereof. **Claim 5** The transcriptional control is regulated by the OCT4 locus, Optionally, the OCT4 locus further comprises a deletion of a proximal enhancer element, Optionally, the OCT4 comprises at least 70% sequence identity or similarity with SEQ ID NO: 1, or a fragment thereof, according to claim 1. **Claim 6** The transgenic mouse according to claim 3, wherein the fluorescent protein is operably linked to the C-terminus of the OCT4 locus. **Claim 7** The transgenic mouse according to claim 1, wherein the germline is selected from semen, oocytes, stem cells, or zygotes. **Claim 8** The transgenic mouse according to claim 1, wherein the transgenic mouse is viable and fertile, and gene expression of the fusion protein is stably integrated into transgenic mouse offspring. **Claim 9** The transgenic mouse according to claim 1, wherein gene expression of the fusion protein in the zygote begins with the development of cells in the 2-cell stage, 3-cell stage, or 4-cell stage. **Claim 10** An embryo expressing an OCT4::EGFP fusion protein, Here, an oocyte is fertilized with semen containing the OCT4::EGFP fusion protein, and the semen is derived from the transgenic mouse according to claim 1, and the embryo.

11. Stem cells that express an OCT4::EGFP fusion protein derived from the transgenic mouse according to claim 1.

12. Germline cells that express an OCT4::EGFP fusion protein derived from the transgenic mouse according to claim 1.

13. A method for producing a transgenic mouse, The method includes the step of microinjecting a zygote with a bacterial artificial chromosome (BAC) construct, Here, the construct contains a reporter gene operably linked to the mouse OCT4 locus, and the zygote is implanted into the reproductive organs of a surrogate mouse, thereby producing a transgenic mouse.

14. The method according to claim 13, wherein the transgenic mouse stably expresses the reporter gene.

15. The locus of the reporter gene is stably inherited via the germline DNA of the transgenic mouse, Optionally, the germline is selected from semen, oocytes, stem cells, or zygotes. The method according to claim 13.

16. The method according to claim 13, wherein the reporter gene encodes a fluorescent protein.

17. The fluorescent protein is selected from green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP), Optionally, the fluorescent protein is selected from GFP or enhanced green fluorescent protein (eGFP) that optionally contains an A206K mutation. The method according to claim 16.

18. The reporter gene contains a nucleic acid sequence encoding a fluorescent protein having at least 70% sequence identity or similarity with SEQ ID NO: 2, or a fragment thereof, Optionally, the reporter gene is operably linked to an OCT4 coding sequence encoding an amino acid sequence having at least 70% sequence identity or similarity with SEQ ID NO: 1, or a fragment thereof. Optionally, the reporter gene and the gene coding sequence are separated by a linker, and the linker comprises an amino acid sequence comprising SGGGGGSGGGGGSGGGGGS (SEQ ID NO: 3). The method according to claim 13, wherein optionally, the reporter gene is operably linked to the C-terminus of the OCT4 locus.

19. The method according to claim 13, wherein the polypeptide comprising the fluorescent protein and OCT4 comprises at least 70% sequence identity or similarity with SEQ ID NO:

4.

20. The method according to claim 13, wherein the construct comprises a nucleic acid sequence comprising at least 70% sequence identity or similarity with SEQ ID NO:

5.

21. The method according to claim 13, wherein the OCT4 locus further comprises a deletion of a proximal enhancer element.

22. The construct mediates the expression of an OCT4::EGFP fusion protein. The method according to claim 13, wherein optionally, the OCT4::EGFP fusion protein is stably integrated into the zygote.

23. A method for evaluating a product for use in assisted reproductive therapy (ART), treatment of a disease, drug screening, or immunomodulation, comprising: (a) obtaining a transgenic embryo comprising stable expression of a fusion protein comprising OCT4; (b) culturing the transgenic embryo; (c) evaluating the expression of the fusion protein; and (d) determining the acceptability or failure of the product A method comprising.

24. The method according to claim 23, wherein the fusion protein is a fluorescent protein fused to OCT4.

25. The fluorescent protein is selected from green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP). The method according to claim 24, wherein optionally, the fluorescent protein is selected from GFP or enhanced green fluorescent protein (eGFP) optionally comprising the A206K mutation.

26. The step of evaluating comprises visualizing the nuclear localization or cytoplasmic localization of the fusion protein. Optionally, the nuclear localization comprises binding of the fusion protein to DNA in the nucleus. Optionally, said evaluating step comprises determining the temporal and spatial expression of said fusion protein, The method according to claim 23, wherein optionally, said evaluating step is performed at the 4-cell stage, 8-cell stage, or blastocyst stage, preferably at the 8-cell stage.

27. Said fusion protein is predominantly localized or expressed in the nucleus at said 4-cell stage, The method according to claim 23, wherein optionally, at least 50%, 60%, 70%, 80%, 90%, 95%, or more of said fusion protein is localized or expressed in the nucleus at said 4-cell stage.

28. Said fusion protein is localized or expressed in the nucleus at said 8-cell stage, The method according to claim 23, wherein optionally, at least 80%, 90%, 95%, or more of said fusion protein is localized or expressed in the nucleus at said 8-cell stage.

29. Said fusion protein is localized or expressed in the inner cell mass (ICM) at said blastocyst stage, The method according to claim 23, wherein optionally, at least 80%, 90%, 95%, or more of said fusion protein is localized or expressed in said ICM at said blastocyst stage.

30. The method according to claim 23, wherein when 1) there is nuclear localization or expression of said fusion protein, and / or 2) there is localization or expression of said fusion protein in said ICM, said product is acceptable.

31. When the nuclear localization or expression of said fusion protein at said 4-cell stage or 8-cell stage is less than 40%, 30%, 20%, 10%, 5%, or 1%, said product is not acceptable, Optionally, when there is no nuclear localization or expression of said fusion protein at said 8-cell stage, said product is not acceptable, Optionally, when the localization or expression of said fusion protein in said ICM is less than 40%, 30%, 20%, 10%, 5%, or 1%, said product is not acceptable, The method according to claim 23, wherein optionally, when there is no localization or expression of said fusion protein in said ICM, said product is not acceptable.

32. The method according to claim 23, wherein said culturing step is in vitro.

33. Said fusion protein is For about 24 hours to about 96 hours of culture, about 24 hours to about 72 hours, about 24 hours to about 48 hours, about 24 hours to about 36 hours, about 36 hours to about 96 hours, about 36 hours to about 72 hours, about 36 hours to about 48 hours, about 48 hours to about 72 hours, or about 48 hours to about 96 hours; or Detectable at about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours of culture, and Optionally, the method according to claim 23, wherein the fusion protein is detectable in the development of cells at the 2-cell stage, 3-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, morula stage, or blastocyst stage.

34. The method according to claim 23, wherein the product is selected from a needle, a catheter, a microtool, a laboratory instrument, a syringe, a tissue culture dish, a tissue culture plate, a pipette tip, a dish, a plate, water, a water purification system, a medium, a medium supplement, or another device or reagent in physical contact with the embryo.

35. The product is a protein or gene related to a disease or embryo development, Optionally, the method according to claim 23, wherein the disease is cancer or an autoimmune disease.

36. Further comprising the step of obtaining one or more embryonic stem cells from the transgenic embryo, and culturing the one or more embryonic stem cells to generate a plurality of embryonic stem cells, Optionally, further comprising the step of incubating the plurality of embryonic stem cells with a drug, the step of evaluating the expression of the fusion protein, and the step of determining the tolerability or failure of the drug, the method according to claim 23.

37. The method according to claim 36, wherein the drug is for use in the treatment of a disease, optionally cancer or an autoimmune disease, or for use in modulating an immune response.

38. A kit comprising the transgenic mouse according to claim 1, the embryo according to claim 10, the stem cells according to claim 11, or the germline cells according to claim 12, optionally including instructions for use.

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