A somatic cell nuclear transfer method and its application

Abstract

The present invention relates to a somatic cell nuclear transfer method and its application. This method uses a haploid embryonic stem cell system to obtain donor somatic cells in which one or more of 26 imprinted genes related to H3K27me3 are knocked out monoallelically. The results show that monoallelic knockout of one or more of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (such as one or more of the four genes Sfmbt2, Jade1, Gab1, and Smoc1) with H3K27me3 makes the imprinted expression patterns of these genes tend to be normal. At the same time, cloning using these donor somatic cells can increase the somatic cell cloning efficiency to 14%, while the somatic cell cloning efficiency of the wild-type control group is 0. In addition, for the cloned animals obtained by this method, the phenomenon of large placenta - large fetus is corrected.

Description

Technical Field

[0001] The present invention relates to the field of animal biotechnology. Specifically, the present invention relates to a somatic cell nuclear transfer method and its application. Background Art

[0002] Somatic cell nuclear transfer technology (SCNT) can reprogram somatic cell nuclei to a totipotent state, and it has great application potential in the fields of animal breeding and regenerative medicine (Rideout et al., 2001). However, the extremely low developmental efficiency of nuclear transfer embryos and the frequently observed developmental abnormalities indicate that there are epigenetic barriers in somatic cell reprogramming (Lu and Zhang, 2015). Among these abnormalities, large offspring syndrome (LOS) is the most common one, and it has been found in cloned cattle, sheep, and mice. This syndrome refers to a group of heterogeneous symptoms, including being too large at birth and severe birth defects (Yang et al., 2007). Progress has been made in identifying and overcoming key epigenetic barriers during nuclear transfer, including inhibiting histone deacetylation, reducing DNA methylation, and removing somatic cell heterogeneous H3K9me3, etc. (Dai et al., 2010; Gao et al., 2018; Kishigami et al., 2006; Matoba et al., 2014). These methods can significantly improve the developmental efficiency of nuclear transfer embryos, but have little effect on the defects of all cloned mammals. Although abnormal imprinting has also been observed in cloned animals, the typical genomic imprinting mediated by DNA methylation is relatively stable and beyond the reprogramming ability of the current oocyte cytoplasm (Humpherys et al., 2014). For example, cloning using primordial germ cells cannot restore the defective imprinting state or produce viable offspring (Inoue et al., 2002; Kamimura, 2014; Lee, 2002; Tucci, 2019). For these reasons, genomic imprinting is generally not considered an epigenetic barrier to nuclear transfer reprogramming (Fulka et al., 2004; Kamimura, 2014). Currently, the donor cells used for cloning are all somatic cells, and the latest research finds that the imprinting patterns of the extra-embryonic tissues of mouse embryos are different from those derived from the fetus, which confirms that there are abnormalities in the development of extra-embryonic tissues cloned with the current conventional donor cells.

[0003] Therefore, the somatic cell nuclear transfer method still needs further research. Summary of the Invention

[0004] This application is made based on the inventor's discovery and understanding of the following facts and problems:

[0005] Successful cloning by somatic cell nuclear transfer requires overcoming the obvious epigenetic barriers. Although the H3K27me3-dependent imprinted genes show differential expression in the embryonic epiblast and extra-embryonic tissues at embryonic day E6.5, genomic imprinting is generally not considered an obstacle to nuclear transfer failure. Based on the above problems, the inventors obtained a method to significantly improve the nuclear transfer efficiency through a large number of experimental explorations. This method uses a haploid embryonic stem cell system to obtain donor somatic cells with monoallelic knockout of one or more of the 26 H3K27me3-related imprinted genes for nuclear transfer. The results showed that monoallelic knockout of one or more of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (such as one or more of the four genes Sfmbt2, Jade1, Gab1, and Smoc1) of H3K27me3 made the imprinted expression patterns of these genes tend to be normal. At the same time, using these donor somatic cells for cloning can increase the somatic cell cloning efficiency to 14%, while the somatic cell cloning efficiency of the wild-type control group is 0. In addition, for the cloned animals obtained by this method, the phenomenon of large placenta-large fetus was corrected.

[0006] Therefore, in the first aspect of the present invention, the present invention provides a modified haploid embryonic stem cell, wherein the haploid embryonic stem cell is a mouse-derived haploid embryonic stem cell, and the haploid embryonic stem cell is knocked out of the genes selected from the following: Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2, and any combination thereof.

[0007] It should be noted that the haploid embryonic stem cell refers to a cell population that contains only one set of chromosomes but has the ability to divide and differentiate similar to normal stem cells.

[0008] In some embodiments, the additional H19 and IG in the haploid embryonic stem cell are knocked out, and optionally, the additional Rasgrf1 is knocked out.

[0009] In some embodiments, additional H19, IG, and Rasgrf1 in the haploid embryonic stem cells are knocked out.

[0010] It should be noted that the method for preparing haploid embryonic stem cells in which H19, IG, and Rasgrf1 are knocked out is described in detail in "Generation of Bimaternal and Bipaternal Mice from Hypomethylated HaploidESCs with Imprinting Region Deletions" (Li et al., 2018, Cell Stem Cell 23, 1–12, November 1, 2018).

[0011] In some embodiments, the haploid embryonic stem cells are knocked out of genes selected from the following: Sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

[0012] In some embodiments, in the haploid embryonic stem cells, Sfmbt2 is knocked out, and optionally, genes selected from the following are knocked out: Jade1, Gab1, Smoc1, and any combination thereof.

[0013] In some embodiments, in the haploid embryonic stem cells, genes or gene combinations selected from the following are knocked out:

[0014] Only Sfmbt2 is knocked out,

[0015] Or, only Sfmbt2 and Jade1 are knocked out, or only Sfmbt2 and Gab1 are knocked out, or only Sfmbt2 and Smoc1 are knocked out,

[0016] Or, only Sfmbt2, Jade1, and Gab1 are knocked out, or only Sfmbt2, Jade1, and Smoc1 are knocked out, or only Sfmbt2, Smoc1, and Gab1 are knocked out,

[0017] Or, Sfmbt2, Jade1, Gab1, and Smoc1 are knocked out simultaneously.

[0018] In a second aspect of the present invention, the present invention provides a method for preparing the aforementioned modified haploid embryonic stem cells, which includes: obtaining the modified haploid embryonic stem cells through gene knockout technology, wherein the haploid embryonic stem cells are mouse-derived haploid embryonic stem cells, and the gene knockout technology knocks out genes selected from the following in the haploid embryonic stem cells: Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2, and any combination thereof.

[0019] In some embodiments, the gene knockout technology further knocks out H19 and IG in the haploid embryonic stem cells, and optionally, further knocks out Rasgrf1.

[0020] In some embodiments, the gene knockout technology knocks out H19, IG, and Rasgrf1 in the haploid embryonic stem cells.

[0021] In some embodiments, the gene knockout technology knocks out genes selected from the following in the haploid embryonic stem cells: Sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

[0022] In some embodiments, the gene knockout technology knocks out Sfmbt2 in the haploid embryonic stem cells, and optionally, knocks out genes selected from the following: Jade1, Gab1, Smoc1, and any combination thereof.

[0023] In some embodiments, the gene knockout technology knocks out the following genes or gene combinations in the haploid embryonic stem cells:

[0024] Only knock out Sfmbt2,

[0025] Or, only knock out Sfmbt2 and Jade1, or only knock out Sfmbt2 and Gab1, or only knock out Sfmbt2 and Smoc1,

[0026] Or, only knock out Sfmbt2, Jade1, and Gab1, or only knock out Sfmbt2, Jade1, and Smoc1, or only knock out Sfmbt2, Smoc1, and Gab1,

[0027] Or, simultaneously knock out Sfmbt2, Jade1, Gab1, and Smoc1.

[0028] In some embodiments, the gene knockout technique is a gene knockout technique using CRISPR.

[0029] In some embodiments, the gene knockout technique is a gene knockout technique using CRISPR-Cas9.

[0030] In some embodiments, using the CRISPR-Cas9 gene knockout technique, two sgRNAs are used to knockout the Gab1 gene, and the sequences of the two sgRNAs are SEQ ID NO:1 and 2 respectively.

[0031] Alternatively, using the CRISPR-Cas9 gene knockout technique, two sgRNAs are used to knockout the Jade1 gene, and the sequences of the two sgRNAs are SEQ ID NO:3 and 4 respectively.

[0032] Alternatively, using the CRISPR-Cas9 gene knockout technique, two sgRNAs are used to knockout the Sfmbt2 gene, and the sequences of the two sgRNAs are SEQ ID NO:5 and 6 respectively.

[0033] Alternatively, using the CRISPR-Cas9 gene knockout technique, two sgRNAs are used to knockout the Smoc1 gene, and the sequences of the two sgRNAs are SEQ ID NO:7 and 8 respectively.

[0034] In some embodiments, the aforementioned modified haploid embryonic stem cells are obtained by the following method:

[0035] For each of the four genes Sfmbt2, Smoc1, Gab1, and Jade1, a pair of sgRNAs is set, and these sgRNAs all target the common exon regions of all transcripts of their respective genes. Subsequently, the plasmids carrying these sgRNAs and Cas9 are transfected into haploid embryonic stem cells with green fluorescent protein expression knocked out of the H19, IG, and Rasgrf1 genes using an electroporator. The cell quantity is at the order of 10^6. After the transfected haploid embryonic stem cells are cultured on the haploid embryonic stem cell medium for two days, the haploid embryonic stem cells with green fluorescent protein are sorted out using a flow cytometer. Preferably, subsequently, the transfected cells are identified using PCR technology, and the haploid embryonic stem cells with complete knockout of these four genes are identified.

[0036] In some embodiments, the method for preparing the aforementioned modified haploid embryonic stem cells further includes: identifying the obtained haploid embryonic stem cells using PCR technology.

[0037] In some embodiments, the primer sequences for Sfmbt2, Smoc1, Gab1, and Jade1 used during the identification are as follows:

[0038] For Gab1, the primer sequences used during the identification are SEQ ID NO:11 and 12,

[0039] Alternatively, for Jade1, the primer sequences used during the identification are SEQ ID NO:13 and 14,

[0040] Alternatively, for Sfmbt2, the primer sequences used during the identification are SEQ ID NO:15 and 16,

[0041] Alternatively, for Smoc1, the primer sequences used during the identification are SEQ ID NO:17 and 18.

[0042] In a third aspect of the present invention, the present invention provides a somatic cell, wherein the somatic cell is derived from a mouse, and the somatic cell is monoallelically knocked out of genes selected from the following: Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2, and any combination thereof.

[0043] In some embodiments, H19 and IG in the somatic cell are additionally monoallelically knocked out, and optionally, Rasgrf1 is additionally monoallelically knocked out.

[0044] In some embodiments, H19, IG, and Rasgrf1 in the somatic cell are additionally monoallelically knocked out.

[0045] In some embodiments, the somatic cell is monoallelically knocked out of genes selected from the following: Sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

[0046] In some embodiments, in the somatic cell, Sfmbt2 is monoallelically knocked out, and optionally, genes selected from the following are monoallelically knocked out: Jade1, Gab1, Smoc1, and any combination thereof.

[0047] In some embodiments, in the somatic cell, genes or gene combinations selected from the following are monoallelically knocked out:

[0048] Only Sfmbt2 is knocked out,

[0049] Alternatively, only Sfmbt2 and Jade1 are knocked out, or only Sfmbt2 and Gab1 are knocked out, or only Sfmbt2 and Smoc1 are knocked out,

[0050] alternatively, only Sfmbt2, Jade1 and Gab1 are knocked out, or only Sfmbt2, Jade1 and Smoc1 are knocked out, or only Sfmbt2, Smoc1 and Gab1 are knocked out,

[0051] alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 are simultaneously knocked out.

[0052] In some embodiments, the somatic cells are fibroblasts.

[0053] In some embodiments, the fibroblasts are fetal or adult fibroblasts (such as tail tip fibroblasts).

[0054] In a fourth aspect of the present invention, the present invention provides a method for preparing the aforementioned somatic cells, which includes:

[0055] (1) providing the aforementioned modified haploid embryonic stem cells,

[0056] (2) injecting the nucleus of the haploid embryonic stem cells into a pre-activated oocyte to obtain a first reconstructed embryo;

[0057] (3) culturing and developing the first reconstructed embryo into a fetus;

[0058] (4) isolating the somatic cells from the fetus.

[0059] In some embodiments, in step (3), the first reconstructed embryo is cultured and developed into a fetus by using a tetraploid blastocyst.

[0060] In some embodiments, the method includes:

[0061] 1) providing the aforementioned modified haploid embryonic stem cells,

[0062] 2) injecting the nucleus of the haploid embryonic stem cells into a pre-activated oocyte to obtain a first reconstructed embryo and culturing it to obtain a first blastocyst,

[0063] 3) isolating the first blastocyst to obtain inner cell mass cells, or an embryonic stem cell line established from the first blastocyst,

[0064] 4) injecting the inner cell mass cells or the embryonic stem cell line into a tetraploid blastocyst and culturing to obtain a second reconstructed embryo,

[0065] 5) Develop the second reconstructed embryo (for example, transplant the second reconstructed embryo into the uterus of a surrogate mother for such development) to obtain a fetus.

[0066] 6) Isolate and obtain the somatic cells from the fetus.

[0067] In some embodiments, the uterus of the surrogate mother is the uterus of a mammalian (preferably non-human mammalian) surrogate mother.

[0068] In some embodiments, the uterus of the surrogate mother is the uterus of a mouse, sheep, cow, pig or monkey.

[0069] It should be noted that oocyte activation means that since mature oocytes are arrested at metaphase II (MII) of meiosis, only under the stimulation of sperm or certain physical and chemical factors can oocytes resume and complete meiosis, and this process is called oocyte activation.

[0070] In some embodiments, the pre-activated oocytes are obtained by the following method: pre-activate the oocytes in a calcium-free CZB culture solution containing SrCl2 at a concentration of 8-12 mM (such as 10 mM) for 20-40 minutes (such as 30 minutes).

[0071] In some embodiments, the pre-activation treatment is carried out in an incubator at an appropriate temperature and with a small amount of CO2. In some preferred embodiments, the pre-activation treatment is carried out in a 37 °C, 5% CO2 incubator.

[0072] It should be noted that the tetraploid blastocyst cannot develop normally, but can form a placenta and can be used to verify whether stem cells have totipotency. The tetraploid blastocyst is an embryo with four sets of chromosomes generated by externally stimulating two diploid embryos at the 2-cell stage.

[0073] In some embodiments, the tetraploid blastocyst is obtained by electrofusion or chemical fusion methods.

[0074] In some embodiments, the tetraploid blastocyst is obtained by the following method: obtain mammalian 2-cell stage embryos, place the 2-cell stage embryos in an embryo fusion solution, and use an electrofusion apparatus to shock the 2-cell stage embryos at a direct current electric field strength of 1-3 kV / cm (such as 2 kV / cm) and a pulse duration of 30-50 μs (such as 40 μs) to obtain the tetraploid blastocyst (such as obtaining the tetraploid blastocyst with two sets of chromosome sets); optionally, place the blastocyst in a culture solution (such as M16 culture solution) for culture (preferably, the culture is carried out in an incubator at an appropriate temperature and with a small amount of CO2, and more preferably, the culture is carried out in a 37 °C, 5% CO2 incubator) for standby.

[0075] In some embodiments, the mammal is a non-human mammal, such as a mouse, sheep, cow, pig or monkey.

[0076] In some embodiments, step 2) is carried out by the following steps:

[0077] Haploid embryonic stem cells in the G0-G1 phase are sorted out using a flow cytometer. Subsequently, the nuclei of the haploid embryonic stem cells with complete knockout of four genes are injected into pre-activated oocytes using a micromanipulator. After injection, the reconstructed embryos are placed in a calcium-free CZB culture solution containing SrCl2 at a concentration of 10 mM and cultured for another 5 hours. After 5 hours, the reconstructed embryos are placed in M16 and cultured continuously. In some preferred embodiments, the culture is carried out in an incubator at an appropriate temperature and with a small amount of CO2. In some more preferred embodiments, the culture is carried out in a 37 °C, 5% CO2 incubator.

[0078] In some embodiments, step 3) is carried out by the following steps:

[0079] The blastocysts obtained in the previous step are placed in 5 mg / ML pronase at 37 °C for 3 minutes to remove the zona pellucida. Subsequently, the blastocysts without the zona pellucida are placed in a DMEM culture solution containing 10% fetal bovine serum and 20% anti-mouse whole serum and incubated in an incubator at an appropriate temperature and with a small amount of CO2 (such as a 37 °C, 5% CO2 incubator) for 3 hours. Subsequently, the blastocysts are washed with a DMEM / fetal bovine serum (10%) culture solution and incubated in 100% mouse serum for 20 min. In some preferred embodiments, before tetraploid complementation, the embryos are gently pipetted to gently remove the TE cells, and the isolated ICM cells are placed in a HER / FBS (GIBCO) culture solution.

[0080] In some embodiments, step 4) is carried out by the following steps:

[0081] The inner cell mass cells obtained in the previous step are selected and injected into the tetraploid blastocysts obtained by the aforementioned method using a micromanipulator, with about 15 inner cell mass cells injected into each blastocyst.

[0082] In some embodiments, step 5) is carried out by the following steps:

[0083] The blastocysts obtained by the aforementioned method are transplanted into the uterus of pseudopregnant mice for development, and then the mice are dissected to obtain fetuses.

[0084] In some embodiments, step 6) is carried out by the following steps:

[0085] Sterilize surgical scissors and forceps by soaking the obtained fetus in alcohol for 2 hours, and remove the fetal head and various organs. Chop the remaining embryo, place the chopped tissue in 2 mL of 0.25% trypsin containing 1 mM ethylenediaminetetraacetic acid, and digest in an incubator at an appropriate temperature (such as 37°C) for 10 minutes. Dilute with an equal volume of DMEM liquid containing 10% fetal bovine serum and penicillin / streptomycin to terminate the digestion reaction, and gently pipette more than 20 times. Dilute the cell suspension with fresh medium, spread it on a 100 mm culture dish, shake well, and culture in an incubator at an appropriate temperature and a small amount of CO2 (such as a 37°C, 5% CO2 incubator) for two days to obtain the somatic cells (such as fetal fibroblasts) with single-allele knockout of the target gene. In some preferred embodiments, the somatic cells (such as fetal fibroblasts) all use the first-generation cells.

[0086] In a fifth aspect of the present invention, the present invention provides the use of the aforementioned somatic cells or the somatic cells obtained by the aforementioned method for nuclear transfer.

[0087] In a sixth aspect of the present invention, the present invention provides a method for somatic cell nuclear transfer, which uses donor somatic cells for nuclear transfer, wherein the donor somatic cells are as described above.

[0088] In some embodiments, the somatic cell nuclear transfer method includes:

[0089] A-1) Inject the donor somatic cells into the cell gap (preferably the perivitelline space) of the target recipient cell (preferably, the target recipient cell is enucleated; more preferably, the recipient cell is an enucleated oocyte), and perform embryo fusion to construct a third reconstructed embryo.

[0090] B-1) Activate the third reconstructed embryo.

[0091] In some embodiments, the somatic cell nuclear transfer method includes:

[0092] A-2) Inject the nucleus of the donor somatic cells into the target recipient cell (preferably, the target recipient cell is enucleated; more preferably, the recipient cell is an enucleated oocyte) to construct a fourth reconstructed embryo.

[0093] B-2) Activate the fourth reconstructed embryo.

[0094] In some embodiments, step A-1) is carried out by the following steps:

[0095] The nucleus of the target recipient cell (such as an oocyte) is removed using a micromanipulator, and then the donor somatic cell (such as a fetal fibroblast) is placed in an inactivated Sendai virus for a few seconds and then taken out. The donor somatic cell carrying the Sendai virus is then injected into the intercellular space (such as the oocyte space) of the enucleated target recipient cell (such as an oocyte) using a Piezo, and then the embryo is placed in an incubator with a suitable temperature and a small amount of CO2 (such as a 37°C, 5% CO2 incubator) until the embryo fuses.

[0096] In some embodiments, step B-1) is performed by the following steps:

[0097] The fused embryos are transferred to culture medium (such as M16 culture medium) for brief culture, then placed in calcium-free CZB culture medium containing 5 mg / mL cytochalasin B and 10 mM strontium chloride for activation for 5-6 hours, and finally cultured in culture medium (such as M16 culture medium).

[0098] In some embodiments, in step A-1) or step B-1), the culturing is performed in an incubator with a suitable temperature and a small amount of CO 2 (eg, a 37° C., 5% CO 2 incubator).

[0099] In some embodiments, the oocyte is obtained by:

[0100] Select female mice of appropriate age, inject pregnant mare serotonin (PMSG), and inject human chorionic gonadotropin (HCG) within 13-17 hours after injection. Perform egg retrieval 13-15 hours after HCG injection. The collected oocytes are washed with Hepes-CZB, then cultured in culture medium (such as M16 culture medium), and placed in an incubator with a suitable temperature and a small amount of CO2 (such as 37°C, 5% CO2 incubator) for use. Granulosa cells are digested and removed with hyaluronidase, and before micromanipulation, oocytes are temporarily stored in CZB culture medium containing 3mg / mL BSA.

[0101] In the seventh aspect of the present invention, the present invention provides the use of the aforementioned modified haploid embryonic stem cells or the aforementioned somatic cells in the preparation of a drug for treating a disease associated with somatic cell nuclear transplantation.

[0102] In some embodiments, the disease associated with somatic cell nuclear transfer is large fetus syndrome.

[0103] In some embodiments, the disease associated with somatic cell nuclear transfer is large placenta and / or large fetus.

[0104] In some embodiments, the disease associated with somatic cell nuclear transfer is gestational diabetes and excessive birth weight of a newborn fetus in a mammal.

[0105] In some embodiments, the large fetus syndrome is large fetus syndrome in mammals (preferably non-human mammals).

[0106] In some embodiments, the mammal is a mouse, sheep, cow, pig or monkey.

[0107] In the eighth aspect of the present invention, the present invention provides a method for treating a disease related to somatic cell nuclear transfer, which includes: performing nuclear transfer using a donor somatic cell, wherein the donor somatic cell is as described above.

[0108] In some embodiments, the disease related to somatic cell nuclear transfer is large fetus syndrome.

[0109] In some embodiments, the disease related to somatic cell nuclear transfer is a large placenta and / or a large fetus.

[0110] In some embodiments, the disease related to somatic cell nuclear transfer is gestational diabetes and the newborn of the mammal has an overweight birth weight.

[0111] In some embodiments, the large fetus syndrome is large fetus syndrome in mammals (preferably non-human mammals).

[0112] In some embodiments, the mammal is a mouse, sheep, cow, pig or monkey.

[0113] In some embodiments, the method for treating a disease related to somatic cell nuclear transfer includes:

[0114] A-1) Injecting the donor somatic cell into the cell gap (preferably the perivitelline space) of a target recipient cell (preferably, the target recipient cell is enucleated; more preferably, the recipient cell is an enucleated oocyte), and performing embryo fusion to construct a third reconstructed embryo,

[0115] B-1) Activating the third reconstructed embryo.

[0116] In some embodiments, the method for treating a disease related to somatic cell nuclear transfer includes:

[0117] A-2) Injecting the nucleus of the donor somatic cell into a target recipient cell (preferably, the target recipient cell is enucleated; more preferably, the recipient cell is an enucleated oocyte) to construct a fourth reconstructed embryo,

[0118] B-2) Activating the fourth reconstructed embryo.

[0119] In a ninth aspect of the present invention, the present invention provides an animal, wherein the animal is monoallelically knocked out of a gene selected from the following: Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2, and any combination thereof.

[0120] In some embodiments, additional H19 and IG in the animal are monoallelically knocked out, and optionally, additional Rasgrf1 is monoallelically knocked out.

[0121] In some embodiments, additional H19, IG, and Rasgrf1 in the animal are monoallelically knocked out.

[0122] In some embodiments, the animal is monoallelically knocked out of a gene selected from the following: Sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

[0123] In some embodiments, in the animal, Sfmbt2 is monoallelically knocked out, and optionally, a gene selected from the following is monoallelically knocked out: Jade1, Gab1, Smoc1, and any combination thereof.

[0124] In some embodiments, in the animal, a gene or gene combination selected from the following is monoallelically knocked out:

[0125] Only Sfmbt2 is knocked out,

[0126] Or, only Sfmbt2 and Jade1 are knocked out, or only Sfmbt2 and Gab1 are knocked out, or only Sfmbt2 and Smoc1 are knocked out,

[0127] Or, only Sfmbt2, Jade1, and Gab1 are knocked out, or only Sfmbt2, Jade1, and Smoc1 are knocked out, or only Sfmbt2, Smoc1, and Gab1 are knocked out,

[0128] Or, Sfmbt2, Jade1, Gab1, and Smoc1 are simultaneously knocked out.

[0129] In some embodiments, the animal is a mammal (preferably a non-human mammal).

[0130] In some embodiments, the mammal is a mouse, sheep, cow, pig, or monkey.

[0131] In a tenth aspect of the present invention, the present invention provides a method for preparing the aforementioned animal, which includes:

[0132] Culturing the activated third reconstructed embryo or the fourth reconstructed embryo obtained by the aforementioned method;

[0133] When the third reconstructed embryo or the fourth reconstructed embryo develops to an appropriate stage (such as developing to 2-cell, or developing to 4-cell, or developing to 8-cell, or developing to more cells), transplant the third reconstructed embryo or the fourth reconstructed embryo into the oviduct or uterus of an animal, and obtain the animal when due.

[0134] In some embodiments, the animal is a mammal (preferably a non-human mammal).

[0135] In some embodiments, the mammal is a mouse, a sheep, a cow, a pig or a monkey.

[0136] Advantageous effects:

[0137] Monoallelic knockout of one or more of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (such as one or more of Sfmbt2, Jade1, Gab1 and Smoc1) H3K27me3 imprinted genes on fibroblasts makes the imprinted expression patterns of these genes tend to be normal. At the same time, using these fibroblasts for cloning can increase the cloning efficiency of fibroblasts to 14%, while the cloning efficiency of wild-type control fibroblast donor cells is 0. In addition, for the cloned animals obtained by this method, the large placenta-large fetus phenomenon has been corrected. Among them, in the single-gene knockout strategies of the four genes Sfmbt2, Jade1, Gab1 and Smoc1, Sfmbt2 is the single gene that most significantly improves the cloning efficiency among these four genes. Description of the drawings

[0138] Figure 1 Indicates the loss of H3K27me3 imprinting in the cloned placenta, where:

[0139] A bar graph showing the relative gene expression levels of autosomal imprinted genes in E10.5 cloned placentas. Fifty imprinted genes detected in the cloned placentas (FPKM > 1) are shown. The expression level of imprinted genes in the placentas of in vitro fertilized fetuses was set to 1. The gene names framed by boxes are H3K27me3 imprinted genes. The horizontal dashed line indicates the imprinted genes overexpressed in the cloned placentas (fold change > 2),

[0140] B bar graph showing the relative expression levels of X chromosome-linked imprinted genes in E10.5 cloned placentas. The gene expression level in the placenta of in vitro fertilized was 1. The horizontal dashed line indicates the imprinted genes overexpressed in the cloned placentas (fold change > 2),

[0141] C bar graph showing the parental allele expression ratios of H3K27me3 imprinted genes in in vitro fertilized and cloned placentas identified by SNPs. The gene names not framed by boxes and those framed by boxes represent classical and H3K27me3 imprinted genes respectively,

[0142] D shows the parental allele expression ratios of H3K27me3 imprinted genes in in vitro fertilized and cloned E19.5 placentas detected by RT-PCR and Sanger sequencing using SNPs as markers,

[0143] E shows the protocol for knocking out 4 H3K27me3 imprinted genes based on haESCs3KO and injecting into MII oocytes,

[0144] F shows on the left: wild-type E9.5 embryos and placentas. On the right: arrested E9.5 fetuses and placentas with 4 H3K27me3 imprinted genes knocked out based on haESCs3KO. The placentas are marked with white triangles. The scale bar is 0.5 mm;

[0145] Figure 2 Showing the preparation of somatic cells with single-allele knockout of 4 genes of H3K27me3 imprinted genes and cloning, where:

[0146] A shows the protocol for preparing Δ4-MEFs3KO cells and using them as nuclear transfer donor cells for cloning. Δ4-embryos3KO were injected into oocytes, and when they developed into blastocysts, their inner cell masses were isolated and then the ICMs were injected into wild-type tetraploid blastocysts and transplanted. Then, MEFs were prepared from Δ4-embryos3KO fetuses at E13.5, and Δ4-NT-mice3KO were cloned using these MEFs as donor cells,

[0147] B represents the immunofluorescence results of H3K27me3 imprinted genes in IVF, WT-NT, and Δ4-NT blastocysts. The red signal is the staining of H3K27me3 imprinted genes. The green signal is the CDX2 staining, and the blue signal is the DAPI staining of DNA. The scale bar is 15 μm,

[0148] C represents the comparison of immunofluorescence of H3K27me3 imprinted genes in TE cells of IVF, WT-NT, and Δ4-NT blastocysts. Each dot represents the protein-to-DAPI intensity ratio of a single TE cell. The solid line represents the mean and standard deviation. "****" indicates p < 0.0001, and ns indicates no significant difference, detected by t-test;

[0149] Figure 3 represents the developmental results of somatic cell cloned embryos carrying single-allele knockout of four H3K27me3 imprinted genes, where:

[0150] A represents the statistics of the implantation rates of wild-type MEFs, Δ4-MEFs, and Δ4-TTFs treated with TSA and untreated,

[0151] B represents a photo of freshly dissected Δ4-NT-mice3KO and placenta in one experiment. The scale bar is 10 mm,

[0152] C represents the comparison of the body weights of wild-type cloned mice and Δ4-NT-mice3KO at the end of the experiment, with in vitro fertilized pups as the control,

[0153] D represents the comparison of the placenta weights of wild-type cloned mice and Δ4-NT-mice3KO at the end of the experiment, with the placenta weights of in vitro fertilized pups as the control,

[0154] E represents a comparison chart of the diameters of wild-type cloned mouse placentas and Δ4-NT-mice3KO placentas. Placentas from in vitro fertilization are used as the control group. "*" represents p < 0.05, "**" represents p < 0.01, "***" represents p < 0.001, "****" represents p < 0.0001, and ns indicates no significant difference, analyzed by t-test,

[0155] F represents a placenta section diagram. The upper part is a HE staining diagram, and the lower part is an immunohistochemical diagram of Lamininα1 antibody. The yellow line in the upper diagram and the red line in the lower diagram respectively mark the fetal blood vessels in the labyrinth layer. The scale bar is 1 mm,

[0156] G represents a bar chart of the proportion of fetal blood vessels in the labyrinth layer. "****" represents p < 0.0001, analyzed by t-test,

[0157] H represents placental sections of IVF, WT-NT, and Δ4-NT-mice3KO. The regions marked with stars above are vascular regions, and the regions marked with stars below are acellular regions in the placenta. The scale bar is 0.15 mm. "*" represents p < 0.05, "**" represents p < 0.01, "***" represents p < 0.001, "****" represents p < 0.0001, ns represents no significant difference, analyzed by t-test,

[0158] I represents a bar graph of the proportion of the acellular region in the labyrinth layer,

[0159] J represents a bar graph of the proportion of fetal blood vessels in the labyrinth layer;

[0160] Figure 4 Indicates that correcting a single H3K27me3 imprinted gene can improve SCNT embryo development to varying degrees, where:

[0161] A represents Δ4-NT-mice3KO and its offspring. The offspring with asterisks are dead pups that inherited the knockout of IG-DMR from the mother,

[0162] B represents the litter size of corrected Δ4-NT-mice3KO, comparable to the WT group,

[0163] C represents a comparison of the brain transcriptomes of Δ4-NT-mice3KO offspring, Sfmbt2Δ / + mice, and WT mice. n = 2. "*" represents p < 0.05, "**" represents p < 0.01, "***" represents p < 0.001, "****" represents p < 0.0001, ns represents no significant difference, analyzed by t-test,

[0164] D represents WT-TTFs, Sfmbt2 Δ / + -TTFs, Jade1 Δ / + -TTFs, Gab1 Δ / + -TTFs, Smoc1 Δ / + Comparison of the implantation rates of -TTFs and Δ3-TTFs,

[0165] E represents a photo of Sfmbt2 Δ / + -NT mice,

[0166] F represents a placental section diagram. The upper part is a HE staining diagram, and the lower part is an immunohistochemical diagram of lamininα1 antibody. The yellow line in the upper figure and the red line in the lower figure mark the fetal blood vessels in the labyrinth layer. The scanned images of each sample are combined into one image by adjusting the scale. Scale bar = 1 mm,

[0167] G represents the proportion of fetal blood vessels in the labyrinth. The data in all figures are presented as mean ± standard deviation. "**" represents p less than 0.01, "***" represents p less than 0.001, ns represents no significant difference, analyzed by t test,

[0168] H represents a schematic diagram of the placenta-specific imprinting pattern of H3K27me3 imprinted genes in fertilized embryos and a diagram of the rescue pattern of low SCNT pup rate and placenta / offspring defects by cloning somatic cells with H3K27me3 heteroplasmic monoallelic knockout.

[0169] All the above graphs and tables show the data as mean ± variance. *T test value p<0.05, **p<0.01, **p<0.001, ***p<0.0001;

[0170] Figure 5 Indicates the analysis of H3K27me3 imprinted gene expression in cloned embryos, Figure 1 Related, among which:

[0171] A is a bar graph showing the relative gene expression levels of autosomal imprinted genes in E10.5 cloned fetuses. The 59 imprinted genes detected in cloned embryos are relatively reliable (FPKM>1). The gene expression level of IVF embryos is set to 1. The gene names framed by boxes are H3K27me3 imprinted genes. The horizontal dotted line indicates the imprinted genes that are overexpressed in cloned embryos (fold change>2).

[0172] B shows the relative expression levels of X-chromosome imprinted genes in E10.5 cloned fetuses. The gene expression level in in vitro fertilized embryos was set to 1.

[0173] C represents the ratio of parental allele expression of H3K27me3 imprinted genes in IVF and cloned embryos identified by SNP (paternal to maternal). The gene names without boxes and the gene names with boxes are classical imprinted genes and H3K27me3 imprinted genes, respectively.

[0174] In D, top: Scheme for knocking out Jade1 exon 3. Middle: sgRNA sequence used for knockout. Bottom: Sanger sequencing results of genome and cDNA, confirming the 320 bp DNA deletion in Jade1 exon 3 (left) and the frameshift mutation in Jade1 mRNA (right),

[0175] In E, top: Smoc1 exon 4 knockout scheme. Middle: sgRNA sequence used for knockout. Bottom: Sanger sequencing results of genome and cDNA, confirming the 639 bp DNA deletion in Smoc1 exon 4 (left) and the frameshift mutation of Smoc1 mRNA (right),

[0176] In F, top: Scheme for knocking out Gab1 exon 3. Middle: sgRNA sequences used for knocking out. Bottom: Sanger sequencing results of genome and cDNA, confirming a 773-bp DNA deletion in Gab1 exon 3 (left) and a frameshift mutation in Gab1 mRNA (right).

[0177] In G, top: Scheme for knocking out Sfmbt2 exons 16 - 17. Middle: sgRNA sequences used for deletion. Bottom: Sanger sequencing results of genome and cDNA, confirming a 3290-bp DNA deletion in Sfmbt2 exons 16 - 17 (left) and a frameshift mutation in Sfmbt2 mRNA (right).

[0178] H shows the immunoblotting results of H3K27me3 imprinted genes in WT haESCs and Δ4-haESCs3KO, and I shows the gene modification map of haploid embryonic stem cells substituting sperm.

[0179] Figure 6 Shows the gene expression and survival curves characterizing Δ4-NT pups, related to Figure 2 where:

[0180] A shows the protein immunoblotting map, showing the expression of H3K27me3 imprinted genes in placentas of expired WT-NT, WT-IVF, and Δ4-NT-mice3KO.

[0181] B shows the comparison of brain transcriptomes between Δ4-NT-mice3KO and WT mice. n = 2.

[0182] C shows the Δ4-TTFs3KO cloned mice and their placentas born in one experiment.

[0183] D shows the survival curves of WT and Δ4-MEFs / TTFs cloned mice.

[0184] Figure 7 Shows the correlation between Δ4 and Xist deletions in mouse SCNT, related to Figure 3 where:

[0185] In A, top: Scheme for knocking out the Xist gene. Middle: sgRNA sequences used for Xist knockout. Bottom: Sanger sequencing results of genome and cDNA, showing a 17301-bp knockout of the Xist gene.

[0186] B shows the comparison of brain transcriptomes between Δ4-NT-mice 3KO and ΔXist / Δ4-NT-mice 3KO n = 2.

[0187] C represents Δ4-NT mice at E19.5 3KO and ΔXist / Δ4-NT-mice 3KO Bar graph of relative expression levels of X chromosome imprinted genes in the placenta. Δ4-NT-mice 3KO The expression level in the placenta was set to 1. n = 2.

[0188] D represents freshly dissected Δ4-TTFs 3KO Photographs of cloned mice and placentas

[0189] E represents the Southern blot hybridization results confirming the monoallelic knockout of Xist Δ / Y in mice

[0190] In F, left panel: Uterus for transplantation of TTF 3KO cloned embryos. Right panel: Xist Δ / Y -TTFs cloned fetuses and implantation sites dissected from a single uterus. Scale bar is 2 mm.

[0191] G represents the ratio of fetuses at different stages to all reconstructed embryos. 2-cell stage embryos were regarded as E1.0; dissected implantation sites were regarded as E5.0; fetuses with white eyes were regarded as having a developmental stage less than E10.5; fetuses with black eyes were regarded as having a developmental stage greater than or equal to E10.5; fetuses with both white and black eyes were also regarded as having a developmental stage less than E10.5. Xist Δ / Y -TTF, n = 5; WT-MEF and △Xist / △4-MEF 3KO , n = 4; △4-MEF 3KO and △4-TTF 3KO , n = 3. "*" represents p < 0.05, "**" represents p < 0.01, "***" represents p < 0.001, "****" represents p < 0.0001, ns represents no significant difference, analyzed by t-test;

[0192] Figure 8 represents the contribution of individual H3K27me3 imprinted genes to the development of cloned placentas, related to Figure 4 wherein:

[0193] A represents the protocol for obtaining TTFs with monoallelic knockout of single H3K27me3 imprinted genes

[0194] B represents Δ4-NT-mice 3KO genotype identification results of surviving offspring

[0195] C represents that different imprinted genes contribute differently to cloning

[0196] D and E represent Sfmbt2 Δ / +Analysis of fetal weight and placental weight of -NT and Δ3-NT,

[0197] F represents WT-NT, Sfmbt2 Δ / + -NT and Δ3-NT cross-sectional views of the vascular region (upper) and acellular discontinuous region (lower) of the placenta of term mice. The regions marked with asterisks are the vascular region and the acellular discontinuous region, respectively. The scale bar is 0.15 mm,

[0198] G represents a bar graph of the area ratio of the acellular region in the labyrinth layer,

[0199] H represents a bar graph of the area ratio of the vascular region in the labyrinth layer. "*" represents p < 0.05, "**" represents p < 0.01, "***" represents p < 0.001, "****" represents p < 0.0001, ns represents no significant difference, by t-test. Specific implementation manners

[0200] The technical solutions in the present invention will be clearly and completely described below through specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0201] Successful cloning by somatic cell nuclear transfer requires overcoming the apparent epigenetic barriers. Although H3K27me3-dependent imprinted genes show differential expression in the epiblast and extraembryonic tissues of embryos at embryonic day E6.5, genomic imprinting is generally not considered an obstacle to nuclear transfer failure. In the present invention, the inventors reported a method that significantly improves the efficiency of nuclear transfer. This method uses a haploid embryonic stem cell system to obtain donor somatic cells in which one or more of the 26 H3K27me3-related imprinted genes are monoallelically knocked out for nuclear transfer. The results showed that monoallelic knockout of one or more of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (such as one or more of the four genes Sfmbt2, Jade1, Gab1, and Smoc1) of H3K27me3 made the imprinted expression patterns of these genes tend to be normal. At the same time, using these fibroblasts for cloning could increase the fibroblast cloning efficiency to 14%, while the cloning efficiency of wild-type control fibroblast donor cells was 0. In addition, for the cloned animals obtained by this method, the phenomenon of large placenta-large fetus was corrected. Among the single-gene knockout strategies of the four genes Sfmbt2, Jade1, Gab1, and Smoc1, Sfmbt2 was the single gene that most significantly improved the cloning efficiency among these four genes. The above results indicate that somatic cells lacking H3K27me3 imprinting are important obstacle factors hindering the post-implantation development of somatic cell nuclear transfer embryos, and correcting the expression patterns of these imprinted genes simultaneously in donor somatic cells can overcome these obstacles of nuclear transfer.

[0202] The present invention will be further explained and illustrated below in conjunction with specific embodiments.

[0203] It should be noted that the sequencing data has been stored in the Genome Sequence Archive of the Beijing Institute of Genomics, Chinese Academy of Sciences, with the accession number CRA002383 (http: / / gsa.big.ac.cn). A list of software used for data analysis and processing can be found in the "Key Resource Table".

[0204] All mouse experiments were performed in accordance with the Guide for the Use of Animals in Research published by the Institute of Zoology, Chinese Academy of Sciences. All mice were housed in the animal facility of the Chinese Academy of Sciences. B6D2F1 (C57BL / 6 × DBA / 2), C57BL / 6, and CD-1 background mice were purchased from Beijing Weitong Lihua Company. PWK / PhJ (stock number 003715) mice and C57BL / 6-Tg (CAGEGFP) 1Osb / J (stock number 003291) mice were purchased from Jackson Laboratory. Male C57BL / 6 mice were used to mate with Δ4-NT mice to obtain offspring carrying isolated non-classical imprinted gene deletions. Female C57BL / 6-Tg (CAGEGFP) 1Osb / J × PWK / PhJ mice were used to provide cumulus cells for nuclear transfer. Female B6D2F1 mice (C57BL / 6×DBA / 2) were used to provide somatic donor cells and oocytes, and CD-1 background mice were used to provide fertilized embryos and pseudopregnant dams.

[0205] The sources of the key reagents involved in the following examples are shown in Table 1.

[0206] Table 1: Sources of key reagents

[0207]

[0208]

[0209]

[0210] In addition, the specific steps of some experimental operations involved in the following embodiments are as follows:

[0211] 1. Oocyte Collection

[0212] Female mice of appropriate age were selected and injected with pregnant mare serotonin (PMSG), and human chorionic gonadotropin (HCG) was injected within 13-17 hours after the injection. Oocyte retrieval was performed 13-15 hours after the HCG injection. The collected oocytes were washed with Hepes-CZB, then cultured in M16 (Sigma) culture medium, and placed in a 37°C, 5% CO2 incubator for culture. Granulosa cells were removed by digestion with hyaluronidase, and oocytes were temporarily stored in CZB culture medium containing 3mg / mL BSA before micromanipulation.

[0213] 2. CRISPR-CAS9 gene editing

[0214] According to Table 2, the inventors constructed a pair of sgRNAs for each of the four genes of each of the present invention patents. These sgRNAs all targeted the common exon regions of all transcripts of their respective genes, and the targeting position of Xist was exons 1-6. Subsequently, the sgRNAs and Cas9 were transfected into 10 6 haESCs using a neon (Invitrogen) electroporator. Two days later, GFP-positive haESCs were sorted by flow cytometry and seeded at low density on feeder cells. After 7 days, single colonies were picked for PCR detection. Different from the method by which the inventors obtained MEFs with quadruple non-classical imprinted gene deletions, the inventors directly transfected the Δ4-MEFs 3KO cell line with the Cas9 and the sgRNA expression plasmid for knocking out Xist to generate ΔXist / Δ4-MEFs 3KO . Subsequently, the transfected Δ4-MEFs 3KO nuclei were injected into enucleated oocytes for cloning, and the reconstructed embryos were transferred into pseudopregnant recipients. E13.5 embryos were taken to obtain a new fibroblast cell line, MEFs. Embryos with single-allele knockout of Xist were screened by PCR and confirmed by Sanger sequencing. Cas9 and the Xist-knockout sgRNA expression plasmid were injected into one-cell stage embryos of C57BL6 mice to obtain Xist Δ / Y mice. The genotypes of the mice were verified by PCR and Sanger sequencing. Xist-knockout female mice were mated with C57BL6 male mice to produce Xist Δ / Y mice. The genotypes of the Xist Δ / Y mice were confirmed by Southern hybridization.

[0215] 3. Intracytoplasmic injection of haploid stem cells (Δ4-haESCs 3KO )

[0216] (1) The method for superovulation and egg collection was as in Method 1. Metaphase II oocytes of the B6D2F1 strain were obtained and placed in an incubator for later use.

[0217] (2) Δ4-haESCs 3KO cells in the G0 or G1 phase were sorted by flow cytometry as donors. Before microinjection, the oocytes were placed in CZB medium containing 10 mM SrCl2 for 30 min. Subsequently, the Δ4-haESCs 3KO cells were respectively injected into the oocytes to construct reconstructed embryos.

[0218] (3) The reconstructed embryos were activated in calcium-free CZB medium containing 10 mM SrCl2 for 5 h. The fully activated embryos were washed twice in M16 and finally cultured in M16 medium and placed in a 37°C, CO2 incubator.

[0219] (4) On the second day, the reconstructed embryos at the 2-cell stage were transferred into pseudopregnant CD-1 female mice or transferred to KSOM culture medium and cultured to blastocysts.

[0220] 4. Mouse embryo immunofluorescence

[0221] All blastocysts were treated with 5 mg / mL pronase (Sigma) for 5 - 10 minutes at 37 °C and 5% CO2 to remove the zona pellucida, transferred to M16 for washing, and then fixed with 4% paraformaldehyde (PFA) for 30 minutes. After washing with PBS, the embryos were transferred to a permeabilization solution containing 200 μl of 0.1% Triton X-100 and permeabilized for 30 min. They were blocked in a 1% BSA blocking solution containing 0.1% Tween-20 and 0.01% Triton X-100 for 1 h. The blocked embryos were transferred to primary antibodies (CDX2 and GAB1 / JADE1 / SMOC1) and incubated at 4 °C overnight. They were washed 3 times with PBS. They were co-incubated with secondary antibodies at room temperature for 1 h. The secondary antibodies used were Alexa 488 donkey anti-mouse and Cyanine3 goat anti-rabbit. The cell nuclei were stained with DAPI at room temperature for 5 min. To compare the extra-embryonic protein levels of H3K27me3-dependent imprinted genes, the relative fluorescence intensities of GAB1, JADE1, and SMOC1 in trophoblast cells from IVF, WT-NT, and Δ4-NT blastocysts were analyzed, and TE cells with positive CDX2 signals were selected for analysis. The ratio of GAB1 / JADE1 / SMOC1 protein to DAPI signal in each TE cell was recorded and analyzed (IMARIS 3D / 4D Visualization & Analysis Software).

[0222] 5. Tetraploid complementation

[0223] The Δ4-3KO embryos were transferred into a pre-warmed pronase solution with a mouth pipette and incubated at 37 °C for 3 min to remove the zona pellucida. The zona-free blastocysts were incubated in DMEM containing 10% fetal bovine serum and 20% anti-mouse whole serum at 37 °C for 3 h. The blastocysts were washed with DMEM / FBS (10%) culture medium and incubated in 100% mouse serum for 20 min. Before tetraploid complementation, the embryos were gently pipetted to gently remove the TE cells, and the isolated ICM cells were placed in HER / FBS (GIBCO) culture medium.

[0224] The acquisition of the preparation of tetraploid blastocysts by tetraploid complementation was referred to the previous description (Zhao et al., 2009). Briefly, 2-cell embryos were retrieved from the oviducts of CD-1 female mice and electrofused to generate tetraploid embryos. Subsequently, 10 to 15 ICM cells were injected into each blastocyst and transferred into the uterus of CD-1 pseudopregnant female mice.

[0225] 6. Preparation of donor cells for somatic cell nuclear transfer

[0226] Fetal fibroblasts (MEFs) were derived from E13.5 Δ4-3KO tetraploid complemented embryos, E13.5 B6D2F1 embryos, and embryos generated by intracytoplasmic injection of haESCs3KO at E13.5. Surgical scissors and forceps were sterilized by soaking in alcohol for 2 h, and the fetal head and various organs were removed. The remaining embryos were minced, and the minced tissues were placed in 2 ml of 0.25% trypsin containing 1 mM ethylenediaminetetraacetic acid and digested in a 37 °C incubator for 10 minutes. The digestion reaction was terminated by diluting with an equal volume of DMEM liquid containing 10% fetal bovine serum and penicillin / streptomycin, and gently pipetted more than 20 times. The cell suspension was diluted with fresh medium, plated on a 100 mm culture dish, shaken well, and cultured in a 37 °C, 5% CO2 incubator for two days. The cells were cryopreserved (passage 0). In all experiments, passage 1 cells were used for MEFs.

[0227] Tail tip fibroblasts (TFFs) were obtained from the tail tips of 6-week-old Δ4-NT-3KO mice, B6D2F1 mice, and mice generated by intracytoplasmic injection of haESCs3KO cells. The excised tail tip tissues were placed at the bottom of a culture dish pre-coated with fibronectin and dried at 25 °C - 27 °C for 10 min. Then, DMEM containing 10% fetal bovine serum was gently added to avoid tissue detachment from the bottom of the dish. The culture dish was placed in an incubator at 37 °C until the fibroblasts divided and converged on the bottom of the dish (passage 0). Cells before passage 3 were used for TTFs in all experiments.

[0228] Cumulus cells were obtained from 8-week-old female mice (B6 / PWK) by superovulation with an injection of 7.5 IU of PMSG and 7.5 IU of hCG. After 15 - 17 h, the oocytes were digested with pre-warmed 300 IU / ml hyaluronidase to collect cumulus cells. Before micromanipulation, the oocytes were stored temporarily in CZB medium containing 3 mg / mL BSA, covered with paraffin oil, and placed in a 37 °C, 5% CO2 incubator. The cumulus cells were washed twice with HEPES-CZB medium and stored at 4 °C for use.

[0229] 7. Somatic cell nuclear transfer and embryo culture

[0230] SCNT used the "one-step method" reported by Zhou et al (2003). For granulosa cell cloning, granulosa cells were injected into enucleated oocytes using Piezo; for fibroblast cloning, inactivated Sendai virus was used to fuse enucleated oocytes and fibroblasts. The manipulated embryos were transferred to M16 culture medium for short-term culture and then activated in an activation solution containing 5 mg / ml cytochalasin B and 10 mM strontium chloride for 5 - 6 h. Then they were cultured in M16 plus TSA for 4 h and finally in M16 culture medium.

[0231] 8. Embryo transfer

[0232] Two-cell stage cloned embryos were transferred into the ampulla of the oviduct of E0.5 pseudopregnant CD-1 female mice; tetraploid blastocyst transfer was to transfer the embryos into the uterus of E2.5 pseudopregnant CD-1 female mice.

[0233] 9. Immunohistochemical staining and histological analysis

[0234] Full-term placentas were fixed in 4% paraformaldehyde, then paraffin-embedded and serially sectioned at a thickness of 4 mm, and then HE staining was performed. For immunohistochemical staining, the sections were rinsed in PBS for 5 minutes, then incubated with blocking buffer (PBS containing 1% BSA and 0.1% Tween-20) at room temperature for 20 minutes, and subsequently co-incubated with the primary antibody for 1 h. After that, the sections were washed three times in PBS containing 0.1% Tween-20 and co-incubated with the secondary antibody for 1 h. Photographs were taken using a panoramic tissue cell analyzer (Leica Aperio VESA8). Placental physiological parameters, including trophoblast area, acellular interruption, and blood vessels, were analyzed using ImageScope (v12.0.1.5027) software.

[0235] 10. RNA extraction and RT-PCR

[0236] Fetal placentas were digested into single cells and sorted by flow cytometry to select green fluorescent protein-positive cells. RNA was extracted from GFP-positive cells using the PureLinkTM RNA Mini Kit. Reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme), and genomic DNA was removed using the gDNA remover provided in the kit before that. Primers designed using Primer Premier5 were used to amplify PCR fragments containing PWK and C57-specific SNP sites. The RT-PCR products were ligated using the pClone007 Blunt Vector Kit (TsingKe). After transformation, they were cultured at 37°C for 12 h, and then single colonies were picked for sequencing analysis.

[0237] 11. Preparation and Data Analysis of RNA-Seq Library

[0238] The placenta was minced and digested with 0.25% trypsin at 37°C. During digestion, it was pipetted several times with a pipette tip to make the digestion more thorough, and the digestion was terminated after 10 - 15 minutes. GFP-positive placental cells were sorted by flow cytometry and subjected to RNA sequencing analysis after collection.

[0239] For transcriptome analysis of mice, the brain tissues of WT mice, Δ4-NT-3KO and self-bred mice were collected for RNA-Seq. Total RNA was extracted from the fetuses using TRIzol, and then reverse transcription polymerase reaction was performed with 1 μg of purified RNA each time.

[0240] RNA-seq library construction and data analysis: RNA purification with two rounds of PolyA tailing was performed for each sample. Paired-end sequencing of 150 bp was carried out using an Illumina HiSeq4000 sequencer. RNA-seq data were analyzed using HISAT2 (version 2.1.0) and Cufflinks (version 2.2.1). Data statistics: The statistics and analysis of some data were performed using GraphPad software. One-way ANOVA was used.

[0241] 12. Western Blot

[0242] To obtain fresh placental cells derived from fetal development, the inventors minced the placenta and digested it with 0.25% trypsin at 37°C and 5% CO2 for 10 - 15 minutes. During digestion, the minced tissue was pipetted up and down several times, and then GFP-positive placental cells were sorted and collected using a flow cytometer. The placental cells or haESCs were dissolved in Pierce IP Lysis Buffer, placed on ice, and protease inhibitor and sodium orthovanadate were added every 30 min. Subsequently, the mixture was centrifuged at 12,000 rpm for 10 min in a 4°C centrifuge, and the supernatant was collected and mixed with 10 mL of sample Buffer (1.25 mL of 0.5 M pH 6.8 Tris-HCl, 2.5 mL glycerin, 2 mL 10% SDS, 200 μL 0.5% bromophenol blue, 3.55 mL H2O, and 0.5 mL β-mercaptoethanol), and then boiled in boiling water for 5 min. The samples were separated by SDS-PAGE using 10 mL of 5% stacking gel (5.7 mL ddH2O, 2.5 mL 1.5 M pH 6.8 Tris-HCl, 1.7 mL 30% acrylamide [acryl:bis acryl = 29:1], 100 μL 10% SDS, 50 μL 10% ammonium persulfate, and 10 μL TEMED) and 10 mL of separating gel (4.1 mL ddH2O, 2.5 mL 1.5 M pH 8.8 Tris-HCl, 3.3 mL 30% acrylamide [acryl:bis acry = 29:1], 100 μL 10% SDS, 50 μL 10% ammonium persulfate, and 5 μL TEMED) at 100 V for 1 h. Then, electrophoresis was carried out at 200 mA and 4°C for 1 h and transferred to nitrocellulose. Subsequently, the membrane was blocked for 1 h at room temperature in TBST buffer (10 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4) containing 3% BSA (Sigma).

[0243] Then it was co-incubated with the primary antibody (diluted in TBST containing 1% BSA) at 4°C overnight. After washing three times with TBST (10 minutes each time), it was co-incubated with the secondary antibody at room temperature for 1 h, and the signal was detected using ECL after washing three times (10 minutes each time).

[0244] 13. Southern blot

[0245] Genomic DNA was extracted from mouse tissues and digested overnight at 37°C with an endonuclease (Takara). The digested DNA fragments were separated on a 0.8% agarose gel and transferred to a positively charged nylon membrane (Roche) for hybridization.

[0246] In addition, the sequences involved in the following examples are shown in Table 2 below.

[0247] Table 2: Primer names and their sequences

[0248]

[0249]

[0250]

[0251] In Example 1, biallelic expression of abnormal H3K27me3 imprinted genes occurred in E19.5 cloned placentas

[0252] To study the specific expression of H3K27me3 imprinted genes, the offspring of mating two inbred mouse strains were used as the research object in this invention.

[0253] Experimental method:

[0254] The female parent was a female mouse of the C57BL / 6-Tg(CAG-EGFP)1Osb / J strain, and the male parent was a male mouse of the PWK / PhJ strain. Somatic cell nuclear transfer was performed using granulosa cells from the F1 generation hybrid mice, and the specific steps were as shown in the above Experimental method 7 (i.e., the part of somatic cell nuclear transfer and embryo culture). At the same time, embryos obtained by in vitro fertilization of oocytes with sperm from C57(GFP+) and PWK were used as controls. Since these imprinted genes have not been detected after E9.5. Therefore, the inventors analyzed E10.5 SCNT and IVF embryos. Fetal-derived placental cells with green fluorescence were sorted using flow cytometry, so that there would be no contamination of maternal cells. Then RNA-seq analysis was performed on these sorted cells.

[0255] Experimental results:

[0256] Compared with the in vitro fertilization control, among the 4 H3K27me3 imprinted genes in cloned placental cells, the expression levels of Sfmbt2, Smoc1, and Jade1 were increased by more than 2-fold ( Figure 1 A). However, only Sfmbt2 was overexpressed in the fetus ( Figure 5 A). Moreover, Xist was also highly expressed 2-fold in the cloned placenta, while the expression of Xist in the cloned fetus was not high ( Figure 1 B and Figure 5B). Many classical imprinted genes, including H19, Meg3, and Cdkn1c, were also abnormally expressed in the cloned fetuses at E10.5 ( Figure 1 A and Figure 5 A).

[0257] Furthermore, the inventors conducted in-depth research on the allele-specific expression by single nucleotide polymorphism (SNP) analysis. The analysis showed that in the cloned fetuses, the expression patterns of classical imprinted genes and H3K27me3 imprinted genes remained unchanged ( Figure 5 C and Table 3). In contrast, in the cloned placentas, the allele-specific expression patterns of 4 H3K27me3 imprinted genes were lost, while the typical imprinted genes were not lost ( Figure 1 C and Table 3).

[0258] Furthermore, the inventors analyzed the status of H3K27me3 imprinted genes in late placentas by isolating GFP-positive cells from in vitro fertilized (IVF) and cloned placentas at E19.5 for reverse transcription PCR and Sanger sequencing analysis. The results showed that these 4 H3K27me3 imprinted genes still maintained the paternally biased expression pattern in the IVF placentas at E19.5, while this pattern disappeared in the cloned placentas ( Figure 1 D). In addition, the H3K27me3 imprinted genes were imprinting-lost in the cloned blastocysts. Therefore, the abnormal biallelic expression of non-classical imprinted genes was maintained throughout the post-implantation development of cloned embryos.

[0259] Table 3: Positions of parental SNPs and counts of reads containing SNPs

[0260]

[0261]

[0262] Continued Table 3

[0263]

[0264] Example 2 Single-allele knockout of 4 H3K27me3 imprinted genes can significantly improve cloning efficiency

[0265] Experimental method:

[0266] The present invention discloses a method for improving the cloning efficiency of mice, as well as cell lines and model mice that can be used to improve the cloning efficiency. The specific steps are as follows:

[0267] 1. Obtain oocytes. Select female mice of appropriate age, inject pregnant mare serotonin (PMSG), and inject human chorionic gonadotropin (HCG) within 13-17 hours after injection. Perform egg retrieval 13-15 hours after HCG injection. The collected oocytes are washed with Hepes-CZB, then cultured in M16 (Sigma) culture medium, and placed in a 37°C, 5% CO2 incubator for culture. Granulosa cells are removed by digestion with hyaluronidase, and before micromanipulation, oocytes are temporarily stored in CZB culture medium containing 3 mg / mL BSA.

[0268] 2. Obtain haploid embryonic stem cells that can replace sperm by knocking out the four genes Sfmbt2, Smoc1, Gab1, and Jade1. According to Table 2, set a pair of sgRNAs for each of the four genes Sfmbt2, Smoc1, Gab1, and Jade1. These sgRNAs target the common exon regions of all transcripts of their respective genes ( Figure 5 DG). The inventors then used an electroporator to electroporate the plasmids carrying these sgRNAs and Cas9 into H19, IG, and Rasgrf1 gene knockout ( Figure 5 1) haploid embryonic stem cells with green fluorescent protein expression. The cell order of magnitude is 10^6 level. After the haploid embryonic stem cells after electroporation were cultured on the haploid embryonic stem cell culture medium for two days, flow cell sorting instrument was used to sort out the haploid embryonic stem cells with green fluorescent protein, and then the cells that had been transfected were identified using PCR technology to identify the haploid embryonic stem cells with these four genes completely knocked out. The identification primers are shown in Table 2.

[0269] 3. Haploid embryonic stem cells with four genes completely knocked out were used as substitute sperm for in vitro fertilization simulation. Haploid embryonic stem cells in the G0-G1 phase were sorted using a flow cytometer. The obtained oocytes were then pre-activated in a 10 mM concentration of Srcl2-containing calcium-free CZB culture medium for 30 minutes. 3KO ) was injected into the pre-activated oocyte using a micromanipulator. After the injection, the reconstructed embryo was placed in a 10 mM Srcl2-containing calcium-free CZB culture medium for 5 hours. After 5 hours, the reconstructed embryo was placed in M16 for further culture. During pre-activation and culture, the embryos were placed in a 37°C, 5% CO2 incubator. When the embryos developed to E3.5 day blastocysts, they were ready for use.

[0270] 4. Obtain tetraploid blastocysts that can replace extra-embryonic tissues. Obtain mouse 2-cell stage embryos, place the 2-cell embryos into mouse embryo fusion medium, and use an electrofusion apparatus to electroshock the embryos at a direct current electric field strength of 2 kV / cm and a pulse duration of 40 μs to obtain tetraploid mouse reconstructed embryos with two sets of chromosome sets. Subsequently, culture the embryos in M16 culture medium until E3.5 days for standby. During culture, the embryos are all placed in an incubator at 37°C and 5% CO2.

[0271] 5. Obtain inner cell mass cells of diploid with four genes knocked out. The inventor places the blastocysts obtained in step 3 into 5 mg / ML pronase in an environment at 37°C for 3 minutes to remove the zona pellucida. Subsequently, place the zona pellucida-removed blastocysts into DMEM culture medium containing 10% fetal bovine serum and 20% 20 anti-mouse whole serum, and incubate in an incubator at 37°C and 5% CO2 for 3 hours. Subsequently, wash the blastocysts with DMEM / fetal bovine serum (10%) culture medium and incubate in 100% mouse serum for 20 min. Before tetraploid compensation, gently pipette the embryos, gently remove the TE cells, and place the isolated ICM cells into HER / FBS (GIBCO) culture medium.

[0272] 6. Obtain fetal fibroblast donor cells with four genes knocked out. Select the inner cell mass cells obtained in step 5, and use a micromanipulator to inject them into the blastocysts in step 4, injecting about 15 inner cell mass cells into each blastocyst. Subsequently, transplant the blastocysts into the uterus of pseudopregnant mice at 2.5 days. When the mice are pregnant until E13.5 days, dissect the mice to obtain fetuses at embryonic stage E13.5 days. Subsequently, soak the fetuses in alcohol for 2 h and use surgical scissors and forceps to remove the fetal heads and various organs. Chop the remaining embryos, place the chopped tissues into 2 ml of 0.25% trypsin containing 1 mM ethylenediaminetetraacetic acid, and digest in an incubator at 37°C for 10 minutes. Dilute with an equal volume of DMEM liquid containing 10% fetal bovine serum and penicillin / streptomycin to terminate the digestion reaction, and gently pipette back and forth more than 20 times. Dilute the cell suspension with fresh medium, spread it on a 100 mm culture dish, shake well, and culture in an incubator at 37°C and 5% CO2 for two days to obtain fetal fibroblasts with four genes knocked out. Freeze the cells (passage 0). In all experiments, the first-generation cells of fetal fibroblasts are used.

[0273] 7. Perform nuclear transfer using donor cells. For the enucleated oocytes, use a micromanipulator to remove the oocyte nucleus. Subsequently, place the fetal fibroblasts obtained in step 6 into inactivated Sendai virus for several seconds and then take them out. Then, use Piezo to inject the donor cells with Sendai virus into the perivitelline space of the enucleated oocytes. Subsequently, place the embryos in an incubator at 37°C with 5% CO2 until embryo fusion. Then, transfer the fused embryos to M16 culture medium for a short-term culture and then place them in calcium-free CZB culture medium containing 5 mg / ml cytochalasin B and 10 mM strontium chloride for activation for 5 - 6 h. Finally, culture them in M16 culture medium. During the culture, the embryos are all placed in an incubator at 37°C with 5% CO2. When the embryo develops to the 2-cell stage, transfer the 2-cell stage cloned embryos into the ampulla of the oviduct of E0.5 pseudopregnant CD-1 female mice. Wait for the mice to be dissected at the due date. When the mouse embryos reach E19.5 days of gestation, dissect the 4 gene knockout due mice that are born.

[0274] This set of process methods is as Figure 2 shown in

[0275] Experimental discussion:

[0276] To address the loss of H3K27me3 imprinting in SCNT embryos, it is necessary to knockout the single alleles of 4 H3K27me3 imprinted genes in somatic cells to restore it. Currently, allele-specific gene knockout can be achieved by targeting DNA sites with SNPs on different alleles or using suboptimal, distance-dependent gene editing strategies. However, it is difficult to practically apply these two methods to knockout four single alleles simultaneously in somatic cells or embryonic stem cells. At the same time, since mice with heterozygous mutations at the Sfmbt2 or Gab1 loci are paternal sterile, it is also impossible to produce mice with single allele deletions of four genes knocked out simultaneously by mating different heterozygous mutant mice.

[0277] Therefore, the inventor used haploid embryonic stem cells (haESCs3KO) developed in the inventor's laboratory that have knocked out three DMRs of H19, IG, and Rasgrf1 as a knockout platform. These haploid embryonic stem cells can maintain their paternal imprinting patterns during passage and manipulation, and when injected into oocytes, their efficiency is similar to that of round spermatids.

[0278] It is worth mentioning that mice produced by injecting haESCs into oocytes can also produce viable offspring through natural mating with wild-type male mice.

[0279] Experimental results:

[0280] Using the CRISPR-Cas9 gene knockout technology, the inventors generated a stem cell line with frameshift mutations in four genes, Sfmbt2, Jade1, Smoc1, and Gab1 (Δ4-haESCs3KO) ( Figure 5 D-H).

[0281] After obtaining these four haploid stem cell lines with H3K27me3 imprinted gene knockouts, the inventors injected Δ4-haESCs3KO into oocytes to obtain reconstructed embryos (Δ4-embryos3KO), which were then transferred into pseudopregnant female mice to observe their developmental capabilities ( Figure 1 E). Through dissection, the inventors found that Δ4-embryos3KO could only develop to E9.5, and its placental development was abnormal, indicating that the monoallelic expression of H3K27me3 imprinted genes plays a crucial role in the development of extra-embryonic tissues ( Figure 1 F and Table 5).

[0282] To obtain somatic cell nuclear transfer donors with monoallelic mutations in H3K27me3 imprinted genes, the inventors used calcium- and magnesium-free CZB medium to isolate the inner cell mass cells of Δ4-embryos3KO blastocysts, and then injected these inner cell mass cells into tetraploid blastocysts derived from wild-type ICR strain mouse embryos. After the operation, the embryos were transferred into the uterus of surrogate mothers ( Figure 2 A). It was found that the reconstructed embryos with tetraploid complementation could develop to term (Table 5). The phenomenon that the developmental ability of Δ4-embryos3KO embryos was rescued by tetraploid blastocysts further demonstrated that extra-embryonic tissue defects restricted their development. Most importantly, at E13.5, the inventors successfully generated fibroblasts (Δ4-MEFs3KO) using the rescued fetuses ( Figure 2 A).

[0283] The inventors used Δ4-MEFs3KO as donor cells for somatic cell nuclear transfer experiments. Prior to this, the inventors' laboratory reported a special pre-implantation embryo culture method: D-culture method. Culturing with M16 medium before the late two-cell stage and with KSOM medium after the late two-cell stage can significantly improve the developmental efficiency of cloned blastocysts. However, compared with the developmental efficiency of cloned embryos using wild-type MEFs, the blastocyst developmental efficiency of cloned embryos (Δ4-NT-embryos3KO) using Δ4-MEFs3KO cells as donor cells did not increase whether the D-culture method was used or not. These results indicate that the knockout of H3K27me3 imprinted genes does not affect the pre-implantation development of cloned embryos.

[0284] By immunofluorescence staining of IVF, WT-SCNT and Δ4-NT-embryos3KO blastocysts, the inventors found that the signal intensities of Gab1, Jade1 and Smoc1 in the ICM of IVF embryos were similar to those in WT SCNT, but their intensities in TE were significantly weaker than those in WT cloned TE ( Figure 2 B). On the other hand, in Δ4-NT-embryos3KO blastocysts, the signals in ICM and TE were weaker than those in WT SCNT, indicating that the protein levels of Gab1, Jade1 and Smoc1 were effectively decreased after monoallelic knockout in SCNT blastocysts ( Figure 2 B). Relative fluorescence intensity analysis also showed that IVF and Δ4-NT-embryos 3KO In blastocyst TEs, the expression levels of H3K27me3 imprinted genes were lower than those in WT SCNT embryos ( Figure 2 C), indicating that the levels of H3K27me3 imprinted genes overexpressed in cloned embryo TE were restored.

[0285] The inventors then performed fallopian tube transplantation of Δ4-NT-embryos3KO at the 2-cell embryo stage. Compared with the control group, the implantation rate of Δ4-NT-embryos3KO was greatly improved and close to the implantation rate of the in vitro fertilization group ( Figure 3 A). In addition, the maturity development efficiency of Δ4-NT-embryos3KO was also greatly improved, reaching 8.5-14.2%, while the maturity development efficiency of wild-type MEFs cloned embryos in the control group was 0%, and the maturity development efficiency of wild-type granulosa cell cloned embryos was only 1.1±0.2%. ( Figure 3 B and Table 4).

[0286] The body weight of Δ4-NT-embryos3KO newborn mice was also similar to that of the control group of in vitro fertilized newborn mice, but was lower than that of cloned mice using wild-type granulosa cells ( Figure 3 C). In addition, the weight of the placenta of the Δ4-NT-mice3KO cloned mice was similar to that of the IVF group, but lower than that of the WT group cloned placenta ( Figure 3 D). The same is true for the diameter of the placenta. The diameter of the Δ4-NT-mice3KO placenta is similar to that of the in vitro fertilized mouse placenta, and is smaller than that of the WT clone group ( Figure 3 E). At the same time, the results of protein immunoblotting showed that the protein expression of H3K27me3 imprinted genes in Δ4-NT-mice3KO placenta also decreased ( Figure 6A), demonstrating that the overexpression of h3k27me3 imprinted genes in cloned placentas can be effectively restored by knockout. Moreover, RNA-seq analysis showed that the brain tissues of wild-type normal mice and Δ4-NT-mice3KO mice had very similar transcriptomes, and the expression of imprinted genes was also relatively similar. Figure 6 B). Hematoxylin and eosin staining and staining with the marker laminin A for fetal endothelium and its associated basement membrane revealed that fetal blood vessels in the labyrinth layer were defective in WT-MEF cloned placentas, while they were largely rescued in Δ4-NT-mice3KO placentas. Figure 3 F-G). In addition, the phenotype of an acellular discontinuous region and extremely low placental vascular density at the maternal-fetal interface was also rescued. Figure 3 H-J) (Georgiades et al., 2001).

[0287] To further investigate the effect of knocking out H3K27me3 imprinted genes in somatic cells on somatic cell nuclear transfer, the inventors selected the tail tips of 6-week-old Δ4-NT-mice3KO mice to prepare tail tip fibroblasts (Δ4-TTFs3KO). Using Δ4-TTFs3KO as donor cells for nuclear transfer, 4.8 ± 1.0% of the embryos developed to term after being transferred into surrogate mother mice, while the birth efficiency of WT TTFs clones was only 0% (Table 4 and Figure 6 C). Although fibroblasts can be used for cloning, the cloning efficiency is very low. Therefore, the cloning efficiencies of Δ4-MEFs3KO and Δ4-TTFs3KO are still quite remarkable.

[0288] The inventors tracked the viability of all Δ4-MEFs / TTFs cloned mice. Among the 23 Δ4 cloned mice, 5 died 21 days after birth, and 18 survived. The overall viability was not different from that of wild-type cloned mice (1 out of 5 died). Figure 6 D). In summary, these results indicate that correcting the abnormal biallelic expression of four h3k27me3 imprinted genes can significantly increase the term rate of SCNT, even for adult-derived fibroblasts.

[0289] Classical literature reports that correcting the abnormal expression of Xist can effectively improve the development efficiency of cloned embryos until term. Therefore, in Δ4-MEFs3KO cells, the inventors co-transfected plasmids of Cas9 and sgRNA for Xist gene knockout. The transfected Δ4-MEFs3KO cells were used as donor cells for nuclear transfer, and then the reconstructed embryos were transferred into surrogate mother mice. At E13.5, the inventors obtained fetuses by dissecting the mother mice, and then detected whether Xist in the fetuses had been knocked out by PCR and Sanger sequencing. The inventors used fetuses with single-allele knockout of Xist to prepare MEFs (ΔXist / Δ4-MEFs3KO)( Figure 7 A). However, when using ΔXist / Δ4-MEFs3KO as donor cells, the cloning efficiency was only 5.2 ± 0.8%, and compared with using Δ4-MEFs3KO, the efficiency of nuclear transfer did not increase further. This indicates that knocking out Xist does not have a synergistic effect with knocking out these 4 H3K27me3 imprinted genes (Table 4 and Figure 7 B–D).

[0290] Restoring the level of Xist did not improve the pre-implantation development of SCNT embryos. The work of the Ogura group demonstrated that there was no statistical difference in the blastocyst rate between cloned embryos injected with Xist-siRNA and embryos injected with control siRNA (Matoba et al., 2011). Meanwhile, when using Sertoli cells or cumulus cells for cloning, the implantation rate of somatic cells with single-allele Xist deletion was not better than that of WT cells. However, the cloning efficiency of granulosa cells and Sertoli cells was very high, and compared with fibroblasts, the implantation rate of both was also high, limiting the room for improvement in the implantation rate by knocking out Xist. To better understand the potential synergistic effect between Δ4 and ΔXist, the inventors used mice with maternal Xist knockout to prepare XistΔ / Y-TTFs as donor cells( Figure 7 E). Similar to the previous results, regardless of whether the D-culture method was used, the blastocyst rate of cloned embryos of XistΔ / Y-TTFs did not increase compared with that of WT-MEF cloning (Table 6). Similar to knocking out the 4 H3K27me3 imprinted genes, knocking out Xist had no obvious effect on the pre-implantation development of cloned embryos.

[0291] Subsequently, the inventors transferred 2-cell stage cloned embryos of XistΔ / Y-TTFs into pseudopregnant mother mice. By dissection, it was found that among the 134 transferred cloned embryos, 65 implantation sites were observed, which was significantly higher than that of the control group where only 5 implantation sites were observed out of 240 transferred embryos (Table 4). This result proves that the implantation rate of cloned embryos can be improved by single-allele knockout of Xist in somatic cells.

[0292] Meanwhile, the inventors detailedly counted the survival rates at five different stages of cloned embryo development (2-cell stage / E1.0; implantation / E5.0; E10.5; ≥E10.5; E19.5), and found that knocking out 4 H3K27me3 imprinted genes and knocking out Xist had very similar contributions to cloned embryos, both of which could improve the implantation rate and post-implantation development rate of cloned embryos ( Figure 7 F-G). However, different from ΔXist / Δ4-MEFs3KO, the inventors did not obtain term mice among 134 Xist-knockout cloned embryos (Table 4). These results provide a possible explanation for the lack of additive effect of Δ4 and ΔXist during nuclear transfer development, because whether it is the X chromosome or autosomes, H3K27me3-dependent imprinted genes have some functional overlap during cloned embryo development, so there is no good additive function in development efficiency when correcting both of them.

[0293] On the other hand, the inventors' results also showed that when using fibroblasts, the ability of correcting H3K27me3 imprinted genes to promote term development was much stronger than that of ΔXist (Table 4).

[0294] Classical literature has reported that cloned embryos treated with the histone deacetylase inhibitor TSA can significantly improve the development efficiency of cloned embryos. However, when the inventors treated the embryos cloned using Δ4-MEFs3KO with TSA, the development efficiency of cloned embryos did not further improve, indicating that there is no synergistic effect between TSA treatment and the knockout of these 4 H3K27me3 imprinted genes (Table 4).

[0295] To identify the fertility of Δ4-NT-mice3KO mice, the inventors mated these cloned mice with wild-type males. Since all Δ4-NT-mice3KO mice are female, knocking out 4 paternally expressed H3K27me3 imprinted genes does not affect their expression and function in offspring.

[0296] Δ4-NT-mice3KO mice can give birth to surviving fetuses ( Figure 4 A). According to Mendel's law, the litter size of each litter of mice was calculated. The number of surviving pups (pups without H19 and IG-DMR knockout) in each litter was multiplied by 4. Compared with the litter size of wild-type female mice, the litter size of Δ4-NT-mice3KO (n = 5) was 6.80 ± 2.71, which was comparable to the litter size of wild-type female mice of 6.14 ± 1.80, suggesting that it has normal fertility ( Figure 4 B). In addition, RNA-seq analysis showed that the offspring of Δ4-NT-mice3KO had a normal transcriptome ( Figure 4 C).

[0297] Example 3 Somatic cells derived from fertilization with single-allele knockout of three-gene / single-gene H3K27me3 imprinted genes can improve placental development and the development of cloned fetuses

[0298] Experimental method:

[0299] The specific steps are basically the same as the experimental method in Example 2, except that: to obtain the donor cells required for this step, we mated the Δ4-NT-mice3KO mice obtained in Example 2 with male wild-type C57BL6 mice to obtain mice with trait segregation of single genes or multiple gene combinations, and used the fibroblasts of these mice for nuclear transfer.

[0300] Experimental discussion:

[0301] To further study the effect of a single non-classical imprinted gene on cloning, the inventors used single-gene single-allele knockout TTFs for cloning (referred to as Sfmbt2Δ / +-TTFs, Jade1Δ / +-TTFs, Gab1Δ / +-TTFs, Smoc1Δ / +-TTFs)( Figure 8 A). Offspring without H19, IG, and Rasgrf1 knockout were used for cloning( Figure 8 B).

[0302] Experimental results:

[0303] The inventors used these tail-tip fibroblasts for nuclear transfer. The results showed that compared with nuclear transfer using wild-type TTFs, the implantation rates of cloned embryos using Sfmbt2Δ / +-TTFs and Jade1Δ / +-TTFs were significantly increased (Table 4 and Figure 4 D). While the implantation rates of cloned embryos using Gab1Δ / +-TTFs and Smoc1Δ / +-TTFs only increased slightly( Figure 4 D), this difference is likely due to the different genetic backgrounds of wild-type mice and Gab1Δ / + / Smoc1 mice, rather than ineffective imprinted gene knockout. Compared with the 0% developmental efficiency of cloning using wild-type TTFs, cloning with Sfmbt2Δ / +-TTFs could obtain full-term cloned mice with an efficiency of 2.4±2.3%, and the developmental efficiency was significantly improved( Figure 4 E and Table 4). Due to the significant effect of Sfmbt2Δ / +-TTFs and Jade1Δ / +-TTFs on post-implantation development( Figure 4 D), the inventors detected the nuclear transfer efficiency of TTFs with single-allele knockout of the two genes Sfmbt2 and Jade1 (Δ2-TTFs)( Figure 8B). 73 embryos were transferred, and no fetuses were born. The implantation rate was 43.8%, similar to that of Sfmbt2Δ / +-TTFs (44.6%) and Jade1Δ / +-TTFs (46.2%) (Table 4). Subsequently, the inventors examined TTFs with single-allele knockout of three genes (Sfmbt2Δ / +, Jade1Δ / +, and Gab1Δ / + [Δ3-TTFs]). The implantation rate of Δ3-TTFs was 53.4%, which was significantly higher than that of wild-type cloned embryos ( Figure 4 D and Table 4). In addition, 4.6 ± 0.6% of Δ3-TTFs cloned embryos reached their efficiency, similar to that of Δ4-TTFs 3KO (4.8 ± 1.0%) (Table 4 and Figure 8 C). Compared with the body weight of WT cloned mice (1.94 ± 0.11 g), the body weights of Sfmbt2Δ / +-NT-mice (1.26 ± 0.06 g) and Δ3-NT-mice (1.27 ± 0.08 g) were significantly reduced ( Figure 8 D). The placental weight of Δ3-NT mice (0.15 ± 0.02 g) was also significantly lower than that of WT cloned placentas ( Figure 8 E). Analysis of the placenta also showed that in the placentas of Sfmbt2Δ / +-NT-mice and Δ3-NT-mice, there was a significant improvement in the proportion of fetal blood vessels in the labyrinth layer ( Figure 4 F-G). However, the defects of too low placental vascular density and too high proportion of acellular discontinuous regions were only improved in the placentas of Δ3-NT-mice, and these were not improved in the placentas of Sfmbt2Δ / +-NT-mice ( Figure 8 F–H). Since genomic imprinting is completely re-established during the development of germ cells in cloned animals, the results of offspring TTFs cloning are also a natural confirmation that knocking out H3K27me3 imprinted genes can improve cloning efficiency.

[0304] Table 4: SCNT embryo development from enucleated oocytes with various donor cells

[0305]

[0306]

[0307] A versus a; B versus b; C versus c; D versus d; F versus f; G versus g: p < 0.01; E versus e: p < 0.05;

[0308] H versus h; I versus i; J versus j; K versus k; L versus l: No significant difference; Δ2 represents monoallelic Sfmbt2, Jade1 double deletions;

[0309] Δ3 represents monoallelic Sfmbt2, Jade1, and Gab1 triple deletions;

[0310] B6 / PWK represents C57BL / 6x PWK / PhJ mice;

[0311] The symbols "-1" and "-2" represent different E13.5 embryos to derive MEFs.

[0312] Table 5: Δ4-embryo under different strategies 3KO Development

[0313]

[0314] Table 6: In vitro development of nuclear transfer embryos with different culture methods

[0315]

[0316] A versus a, B versus b, D versus d, E versus e: no significant difference.

[0317] C versus c: p<0.05.

[0318] Thoughts and discussions

[0319] Large fetuses and placentas have been observed in somatic cell cloned mice (Tamashiro et al., 2002), sheep (Fletcher et al., 2007), and cattle (Smith et al., 2012). It has been reported that placental overgrowth precedes fetal overgrowth, so it is very likely that the large placenta causes the large fetus. In the present invention, the inventors demonstrated that biallelically expressed h3k27me3 imprinted genes significantly hampered the development of SCNT placentas. Since h3k27me3 imprinting is naturally absent in somatic cell lines of epiblast origin, if somatic cells are used as donors, h3k27me3 imprinting disorders may affect all cloned placentas ( Figure 4 H).

[0320] After the monoallelic deletion of h3k27me3 imprinted genes in somatic cell donor cells, placental weight, the proportion of fetal-derived blood vessels in the labyrinth layer, blood vessel density, and the acellular discontinuous region of the placenta were well improved. Moreover, the body weight of cloned mice was significantly restored. Since imprint loss specifically occurs in the placenta, this improvement not only proves that abnormal h3k27me3 imprinting in the placenta is the cause of large fetuses, but also supports the hypothesis that abnormal placental genes in cloned animals may lead to the LOS phenotype.

[0321] The abnormal h3k27me3 imprinting of SCNT blastocysts was first discovered by Matoba et al. (Matoba et al., 2018). However, the developmental significance of this discovery has not been determined until this application. Different from classical imprinted genes, whose loss in cloning is relatively random, the loss of non-classical imprinted gene imprinting is relatively fixed ( Figure 1 C and Figure 5 C). This difference makes H3K27me3 a new type of abnormal imprinting affecting somatic cell cloned embryos, which can explain why the expiration rate increases after the deletion of h3k27me3 imprinted genes.

[0322] Since no offspring TTFs lacking H19, IG, or Rasgrf1 deletions were used for cloning, the observed effects were only due to the deletion of h3k27me3 imprinting. The inventors found that single-gene monoallelic knockout (Sfmbt2Δ / +) could very effectively improve placental defects and increase the cloning birth rate (although to a lesser extent than 4KO). Combining gene editing with the epigenetic erasure technology targeting SNPs, the method of knocking out h3k27me3 imprinting may also have broader applications.

[0323] It has been reported that Xist presents an h3k27me3 imprinting pattern, is imprinted and silent in extra-embryonic tissues, and loses imprinting in the epiblast. Like the four knocked-out h3k27me3 imprinted genes, the inventors found that Xist was overexpressed 2-fold in SCNT placentas ( Figure 1B). Notably, the deletion of the Xist monoallele in Sertoli cells significantly increased the cloning rate, from 1.6% to 15.4%. These results suggest that the loss of Xist imprinting is another H3K27me3-dependent epigenetic barrier in SCNT. Recently, some putative autosomal non-classical imprinted genes have been found in human embryos, suggesting that H3K27me3-dependent imprinting may also be conserved in mammals that are evolutionarily distant. However, it has recently been reported that H3K27me3 modifications are widely erased in human embryos at the 4-8 cell stage, and Xist is not spared either. This controversial result suggests that there may be no H3K27me3 imprinting in humans. In addition, the maternal-specific Xist imprinting is not conserved in many species, which may limit the application of Xist knockout in improving animal cloning efficiency.

[0324] Fetal or adult fibroblasts have been widely used in the cloning of important large animals, including sheep (Schnieke et al., 1997), cattle (Cibelli et al., 1998), pigs (Onishi et al., 2000) and monkeys (Liu et al., 2018), etc. However, compared with other donor cell types, the cloning efficiency of mouse fibroblasts is relatively low. The inventors' research results show that after the deletion of H3K27me3-dependent imprinted genes, the cloning efficiency of both fetal and adult fibroblasts is significantly improved. More importantly, the improvement of the Δ4 or Sfmbt2 knockout on the expiration rate is significantly higher than that of the ΔXist fibroblasts. The inventors not only demonstrated that the deletion of H3K27me3-dependent imprinting is an inherent post-implantation epigenetic barrier in SCNT-mediated cloning, leading to large placentas and offspring defects, but also confirmed that correcting H3K27me3 imprinting may be an effective way to improve animal cloning efficiency.

[0325] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. SEQUENCE LISTING <110> Institute of Zoology, Chinese Academy of Sciences <120> A Somatic Cell Nuclear Transfer Method and Its Application <130> IDC200253 <160> 30 <170> PatentIn version 3.3 <210> 1 <211> 20 <212> DNA <213> Artificial <220> <223> Gab1 #1 <400> 1 agattggcct tgaactggta 20 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> Gab1 #2 <400> 2 gagtctcgaa gtctttgtta 20 <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> Jade1#1 <400> 3 ccaactcttt acttggatgc 20 <210> 4 <211> 20 <212> DNA <213> Artificial <220> <223> Jade1#2 <400> 4 cactttatac cccggcaaga 20 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> Sfmbt2#1 <400> 5 agtcatcccg gaccaccacg 20 <210> 6 <211> 20 <212> DNA <213> Artificial <220> <223> Sfmbt2#2 <400> 6 ataactagaa gagtccaaag 20 <210> 7 <211> 20 <212> DNA <213> Artificial <220> <223> Smoc1#1 <400> 7 tcacacgtag aacagtccgg 20 <210> 8 <211> 20 <212> DNA <213> Artificial <220> <223> Smoc1#2 <400> 8 tgccatggac acagggaagg 20 <210> 9 <211> 20 <212> DNA <213> Artificial <220> <223> Xist#1 <400> 9 ctgatccgcg gcgctgaagg 20 <210> 10 <211> 20 <212> DNA <213> Artificial <220> <223> Xist#2 <400> 10 ggatttagtt ccgtctcaag 20 <210> 11 <211> 20 <212> DNA <213> Artificial <220> <223> Gab1 F <400> 11 atctcctcag gccccatctt 20 <210> 12 <211> 20 <212> DNA <213> Artificial <220> <223> Gab1 R <400> 12 agtcacagct tggagtcagc 20 <210> 13 <211> 20 <212> DNA <213> Artificial <220> <223> Jade1 F <400> 13 gggtgatggc actgcaggtt 20 <210> 14 <211> 20 <212> DNA <213> Artificial <220> <223> Jade1 R <400> 14 ccctaagcaa ccgaggctgg 20 <210> 15 <211> 23 <212> DNA <213> Artificial <220> <223> Sfmbt2 F <400> 15 agttggagct tctggtgagc ctt 23 <210> 16 <211> 21 <212> DNA <213> Artificial <220> <223> Sfmbt2 R <400> 16 gggtcagtgg ggaggcggat t 21 <210> 17 <211> 23 <212> DNA <213> Artificial <220> <223> Smoc1 F <400> 17 cccagacggc taggtctgac tcg 23 <210> 18 <211> 22 <212> DNA <213> Artificial <220> <223> Smoc1 R <400> 18 gggtgaagtg tggggctttg tg 22 <210> 19 <211> 19 <212> DNA <213> Artificial <220> <223> Xist F <400> 19 tccaagacgc ggagcgata 19 <210> 20 <211> 20 <212> DNA <213> Artificial <220> <223> Xist R <400> 20 cggccactac tatgagcagg 20 <210> 21 <211> 20 <212> DNA <213> Artificial <220> <223> Gab1 F <400> 21 cgggtgaaga gctggatgag 20 <210> 22 <211> 20 <212> DNA <213> Artificial <220> <223> Gab1 R <400> 22 gtctaaaggt gccggcttga 20 <210> 23 <211> 20 <212> DNA <213> Artificial <220> <223> Jade1 F <400> 23 acttacatgg tgacccgcag 20 <210> 24 <211> 20 <212> DNA <213> Artificial <220> <223> Jade1 R <400> 24 ctttctgaga cctccagccc 20 <210> 25 <211> 20 <212> DNA <213> Artificial <220> <223> Sfmbt2 F <400> 25 atagagctgc aggactccca 20 <210> 26 <211> 20 <212> DNA <213> Artificial <220> <223> Sfmbt2 R <400> 26 cttggtcact gatgcagggt 20 <210> 27 <211> 25 <212> DNA <213> Artificial <220> <223> Smoc1 F <400> 27 ggaagaaaag tctcctttcg tttct 25 <210> 28 <211> 19 <212> DNA <213> Artificial <220> <223> Smoc1 R <400> 28 gcgccctgtg tctactagg 19 <210> 29 <211> 19 <212> DNA <213> Artificial <220> <223> ΔXist F <400> 29 tccaagacgc ggagcgata 19 <210> 30 <211> 20 <212> DNA <213> Artificial <220> <223> ΔXist R <400> 30 cggccactac tatgagcagg 20

Claims

1. A method for preparing modified haploid embryonic stem cells, comprising: Obtaining modified haploid embryonic stem cells through gene knockout technology, wherein the haploid embryonic stem cells haESCs3KO with the three DMRs of H19, IG, and Rasgrf1 knocked out are used as a knockout platform, and the gene knockout technology simultaneously knocks out Sfmbt2, Jade1, Gab1, and Smoc1 in the haploid embryonic stem cells, and the haploid embryonic stem cells are mouse-derived haploid embryonic stem cells.

2. The method according to claim 1, wherein The gene knockout technology is a gene knockout technology using CRISPR.

3. A somatic cell, wherein, In the somatic cells, H19, IG, and Rasgrf1 are monoallelically knocked out, and in the somatic cells, a gene or gene combination selected from the following is monoallelically knocked out: Only Sfmbt2 is knocked out, Or, only Jade1 is knocked out, Or, only Gab1 is knocked out, Or, only Smoc1 is knocked out, Or, only Sfmbt2 and Jade1 are knocked out, Or, only Sfmbt2, Jade1, and Gab1 are knocked out, Or, Sfmbt2, Jade1, Gab1, and Smoc1 are simultaneously knocked out; The somatic cells are derived from mice; The somatic cells are fibroblasts.

4. The somatic cell according to claim 3, wherein, The fibroblasts are fetal or adult fibroblasts.

5. The somatic cell according to claim 4, wherein, The adult fibroblasts are tail tip fibroblasts.

6. A method for preparing the somatic cells according to any one of claims 3-5, comprising: (1) Providing modified haploid embryonic stem cells, wherein Sfmbt2, Jade1, Gab1, and Smoc1 are simultaneously knocked out in the haploid embryonic stem cells, and the haploid embryonic stem cells haESCs3KO with the three DMRs of H19, IG, and Rasgrf1 knocked out are used as a knockout platform, and the haploid embryonic stem cells are mouse-derived haploid embryonic stem cells, (2) Injecting the nucleus of the haploid embryonic stem cells into a pre-activated oocyte to obtain a first reconstructed embryo; (3) Culturing and developing the first reconstructed embryo into a fetus; (4) Isolating the somatic cells from the fetus; In step (3), the first reconstructed embryo is cultured and developed into a fetus by using a tetraploid blastocyst.

7. The method according to claim 6, wherein The method includes: 1) Providing the modified haploid embryonic stem cells, 2) Injecting the nucleus of the haploid embryonic stem cells into a pre-activated oocyte to obtain a first reconstructed embryo and culturing it to obtain a first blastocyst, 3) Isolating the first blastocyst to obtain inner cell mass cells or an embryonic stem cell line established from the first blastocyst, 4) Injecting the inner cell mass cells or the embryonic stem cell line into a tetraploid blastocyst and culturing to obtain a second reconstructed embryo, 5) Developing the second reconstructed embryo to obtain a fetus, 6) Isolating the somatic cells from the fetus.

8. The method according to claim 7, wherein The development of the second reconstructed embryo is to transplant the second reconstructed embryo into the uterus of a surrogate mother for the development, and the uterus of the surrogate mother is the uterus of a mouse mother.

9. The method according to claim 6, wherein The tetraploid blastocyst is obtained by an electrofusion or chemical fusion method. Use of the somatic cells according to any one of claims 3-5 or the somatic cells obtained by the method according to any one of claims 6-9 for nuclear transfer, said use being for non-therapeutic purposes.

11. A method for somatic cell nuclear transfer, which uses donor somatic cells for nuclear transfer, wherein, The donor somatic cells are defined as in any one of claims 3-5, and the method is for non-therapeutic purposes.

12. The method according to claim 11, wherein, The method comprises: A-1) injecting the donor somatic cells into the intercellular space of a target recipient cell and performing embryo fusion to construct a third reconstructed embryo; B-1) activating the third reconstructed embryo.

13. The method according to claim 12, wherein, The target recipient cell is enucleated.

14. The method according to claim 12, wherein, The recipient cell is an enucleated oocyte.

15. The method according to claim 12, wherein, The intercellular space is the perivitelline space.

16. The method according to claim 11, wherein, The method comprises: A-2) injecting the nucleus of the donor somatic cells into a target recipient cell to construct a fourth reconstructed embryo; B-2) activating the fourth reconstructed embryo.

17. The method according to claim 16, wherein The target recipient cell is enucleated.

18. The method according to claim 16, wherein, The recipient cell is an enucleated oocyte.

19. A method for preparing an animal, which comprises: culturing the activated third reconstructed embryo obtained by the method according to claim 12 or the activated fourth reconstructed embryo obtained by the method according to claim 16; when the third reconstructed embryo or the fourth reconstructed embryo develops to an appropriate stage, transplanting the third reconstructed embryo or the fourth reconstructed embryo into the oviduct or uterus of an animal, and obtaining the animal at term, the animal being a mouse.

20. The method according to claim 19, wherein, The appropriate stage is development to the 2-cell stage, or development to the 4-cell stage, or development to the 8-cell stage, or development to more cells.

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