CN113801851A

CN113801851A - Somatic cell nuclear transplantation method and application thereof

Info

Publication number: CN113801851A
Application number: CN202110666994.0A
Authority: CN
Inventors: 周琪; 李伟; 王乐韵; 李治琨; 王立宾
Original assignee: Institute of Zoology of CAS
Current assignee: Institute of Zoology of CAS
Priority date: 2020-06-17
Filing date: 2021-06-16
Publication date: 2021-12-17

Abstract

The invention relates to a somatic cell nuclear transplantation method and application thereof. The method utilizes a haploid embryonic stem cell system to obtain a single allele and simultaneously knock out one or more donor somatic cells in 26H 3K27me 3-related imprinted genes for nuclear transplantation. The results show that single-allele knockout of imprinted genes of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, tl 3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (one or more of four such as Sfmbt2, Jade1, Gab1 and Smoc1) H3K27me3 makes the expression patterns of these imprinted genes tend to be normal. Meanwhile, the somatic cell cloning efficiency can be improved to 14% by using the donor somatic cell for cloning, and the somatic cell cloning efficiency of a wild-type control group is 0. In addition, the cloned animal obtained by the method has corrected large placenta-large fetus phenomenon.

Description

Somatic cell nuclear transplantation method and application thereof

Technical Field

The invention relates to the technical field of animal biology, in particular to a somatic cell nuclear transfer method and application thereof.

Background

Somatic cell nuclear transfer technology (SCNT) can reprogram somatic cell nuclei to a totipotent state, which has great application potential in the fields of animal breeding and regenerative medicine (Rideout et al, 2001). However, the extremely low developmental efficiency and often observed dysplasia of nuclear transfer embryos suggest that epigenetic disorders exist for somatic reprogramming (Lu and Zhang, 2015). Among these abnormalities, the fetal syndrome (LOS) is the most common one, found in cloned cows, sheep and mice. This syndrome refers to a heterogeneous group of symptoms, including birth-time oversized and severe birth defects (Yang et al, 2007). Research has been advanced in identifying and overcoming key epigenetic disorders in the nuclear transfer process, including H3K9me3 and the like (Dai et al, 2010; Gao et al; 2018; Kishigami et al, 2006; Matoba et al, 2014) that inhibit histone deacetylation, reduce DNA methylation, and remove somatic heterogeneity, all of which significantly improve the developmental efficiency of nuclear transfer embryos, but have little effect on defects in all cloned mammals. Although imprinting abnormalities were also observed in cloned animals, typical genomic imprinting mediated by DNA methylation was relatively stable, exceeding the current ability of oocytes to reprogram (Humpherys et al, 2014). For example, cloning with primordial germ cells does not restore the defective imprinting state or produce viable pups (Inoue et al, 2002; Kamimura, 2014; Lee, 2002; Tucci, 2019). For the above reasons, genomic imprinting is not generally considered an epigenetic barrier to nuclear transfer reprogramming (Fulka et al, 2004; Kamimura, 2014). The donor cells cloned at present are somatic cells, and the recent research finds that the embryo imprinting pattern of mouse extraembryonic tissues is different from that of a carcass source, which proves that the tissue development of the donor cells cloned by the conventional donor cells at present is abnormal.

Therefore, somatic cell nuclear transfer methods need further investigation.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

successful cloning by somatic cell nuclear transfer requires overcoming a significant epigenetic barrier. Although H3K27me 3-dependent imprinted genes may exhibit differential expression in embryonic epiblast and extraembryonic tissues at embryonic stage E6.5 days, genomic imprinting is not generally considered to be a barrier to nuclear transfer failure. Based on the above problems, the inventors have conducted extensive experimental studies to obtain a method for significantly improving the efficiency of nuclear transfer, which uses a haploid embryonic stem cell system to obtain donor somatic cells with single allele knockout of one or more of 26H 3K27me 3-related imprinted genes for nuclear transfer. The results show that single-allele knockout of imprinted genes of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, tl 3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (one or more of four such as Sfmbt2, Jade1, Gab1 and Smoc1) H3K27me3 makes the expression patterns of these imprinted genes tend to be normal. Meanwhile, the somatic cell cloning efficiency can be improved to 14% by using the donor somatic cell for cloning, and the somatic cell cloning efficiency of a wild-type control group is 0. In addition, the cloned animal obtained by the method has corrected large placenta-large fetus phenomenon.

To this end, in a first aspect of the present invention, the present invention provides a modified haploid embryonic stem cell, wherein the haploid embryonic stem cell is knocked out for a gene selected from the group consisting 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, and any combination thereof.

It should be noted that the haploid embryonic stem cell refers to a cell population which only contains one set of chromosomes but has the division and differentiation capacity similar to that of a normal stem cell.

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

In some embodiments, additional H19, IG, and Rasgrf1 are knocked out in the haploid embryonic stem cell.

Note that the methods for preparing H19, IG and Rasgrf1 knocked-out Haploid embryonic Stem cells are described in the Generation of Bimatological and Bispecific Mice from methylated haploids with expression details (Li et al, 2018, Cell Stem Cell 23, 1-12, November 1,2018).

In some embodiments, the haploid embryonic stem cell is knocked out for a gene selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, the haploid embryonic stem cell is one in which Sfmbt2 is knocked out, and optionally, a gene selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, in the haploid embryonic stem cell, a gene or combination of genes selected from the group consisting of:

only Sfmbt2 is knocked out,

alternatively, only Sfmbt 2and Jade1, or only Sfmbt 2and Gab1, or only Sfmbt 2and Smoc1,

alternatively, only Sfmbt2, Jade1 and Gab1, or only Sfmbt2, Jade1 and Smoc1, or only Sfmbt2, Smoc1 and Gab1,

alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 were simultaneously knocked out.

In a second aspect of the present invention, the present invention provides a method for preparing the aforementioned modified haploid embryonic stem cell, comprising: obtaining the modified haploid embryonic stem cell by a gene knockout technique, wherein the gene knockout technique knocks down a gene in the haploid embryonic stem cell selected from the group consisting 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, and any combination thereof.

In some embodiments, the gene knockout technique knocks out additional H19 and IG in the haploid embryonic stem cell, and optionally, knockouts additional Rasgrf 1.

In some embodiments, the gene knockout technique knocks out additional H19, IG, and Rasgrf1 in a haploid embryonic stem cell.

In some embodiments, the gene knockout technique knocks out a gene in a haploid embryonic stem cell selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, the gene knockout technique knocks out Sfmbt2 in a haploid embryonic stem cell, and optionally, knockouts selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, the gene knockout technique knocks out a gene or combination of genes in the haploid embryonic stem cell selected from the group consisting of:

only Sfmbt2 is knocked out,

alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 were knocked out simultaneously.

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

In some embodiments, the knockout technique is a knockout technique using CRISPR-Cas 9.

In some embodiments, the Gab1 gene is knocked out using two sgRNAs with sequences of SEQ ID NOS: 1 and 2, respectively, using a CRISPR-Cas9 gene knock-out technique,

or knocking out the Jade1 gene by using two sgRNAs with sequences of SEQ ID NO:3 and 4 respectively by using CRISPR-Cas9 gene knockout technology,

or knocking out the Sfmbt2 gene by using two sgRNAs with sequences of SEQ ID NO:5 and 6 respectively by using CRISPR-Cas9 gene knocking-out technology,

alternatively, the Smoc1 gene was knocked out using two sgRNAs with sequences of SEQ ID NOS: 7 and 8, respectively, using CRISPR-Cas9 gene knock-out technology.

In some embodiments, the modified haploid embryonic stem cell is obtained by a method comprising:

a pair of sgRNAs is arranged aiming at each of four genes of Sfmbt2, Smoc1, Gab1 and Jade1, and the sgRNAs target universal exon regions of all transcripts of the respective genes. Plasmids carrying these sgrnas and Cas9 were then electrotransferred to H19, IG, and Rasgrf1 knockout haploid embryonic stem cells with green fluorescent protein expression using an electrotransfer instrument. The order of cells is 10^ 6. And (3) after the electrically transformed haploid embryonic stem cells are cultured on a haploid embryonic stem cell culture medium for two days, sorting the haploid embryonic stem cells with green fluorescent protein by using a flow cytometry sorter. Preferably, the cells that have been transfected are then identified by PCR techniques to identify haploid embryonic stem cells with complete knockouts of these four genes.

In some embodiments, the method of making the aforementioned modified haploid embryonic stem cell further comprises: and identifying the obtained haploid embryonic stem cells by utilizing a PCR (polymerase chain reaction) technology.

In some embodiments, the primer sequences employed in the identification for Sfmbt2, Smoc1, Gab1, and Jade1, respectively, are:

for Gab1, the primer sequences used for identification are SEQ ID NOS: 11 and 12,

alternatively, for Jade1, the primer sequences used for the identification are SEQ ID NO 13 and 14,

alternatively, for Sfmbt2, the primer sequences used for the identification are SEQ ID NO 15 and 16,

alternatively, for Smoc1, the primer sequences used for the identification were SEQ ID NO 17 and 18.

In a third aspect of the invention, the invention provides a somatic cell, wherein the somatic cell has been single-allele knocked out for a gene selected from the group consisting 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, and any combination thereof.

In some embodiments, additional H19 and IG are single-allele knockouts in the somatic cell, and optionally, additional Rasgrf1 is single-allele knockouts.

In some embodiments, additional H19, IG, and Rasgrf1 are single-allele knockouts in the somatic cell.

In some embodiments, the somatic cell is single-allele knocked-out of a gene selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, in the somatic cell, Sfmbt2 is single-allele knocked out, and optionally, a gene selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, in the somatic cell, a gene or combination of genes selected from the group consisting of:

only Sfmbt2 is knocked out,

alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 were simultaneously knocked out.

In some embodiments, the somatic cell is derived from a mammal (preferably a non-human mammal).

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

In some embodiments, the somatic cell is a fibroblast.

In some embodiments, the fibroblast is a fetal or adult fibroblast (e.g., a tip fibroblast).

In a fourth aspect of the present invention, the present invention provides a method for preparing the aforementioned somatic cell, comprising:

(1) providing the modified haploid embryonic stem cell as described above,

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

(3) culturing and developing the first reconstituted embryo into a fetus;

(4) isolating the somatic cells from the fetus.

In some embodiments, in step (3), the first reconstituted embryo is cultured to develop into a fetus by using a tetraploid blastocyst.

In some embodiments, the method comprises:

1) providing the modified haploid embryonic stem cell as described above,

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

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

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

5) developing the second reconstructed embryo (e.g., the development is performed by transferring the second reconstructed embryo into a uterus of a surrogate mother), obtaining a fetus,

6) isolating the somatic cells from the fetus.

In some embodiments, the surrogate mother uterus is a mammalian (preferably non-human mammal) surrogate mother uterus.

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

It should be noted that, the oocyte activation refers to that, since the mature oocyte is arrested in the second meiotic metaphase (MII phase), the oocyte can recover and complete meiosis only under the stimulation of sperm or some physicochemical factors, and the process is called oocyte activation.

In some embodiments, the pre-activated oocyte is obtained by: the oocytes are incubated at a concentration of 8-12mM (e.g., 10mM) containing Srcl₂Pre-activation treatment in calcium-free CZB medium is carried out for 20-40 min (e.g. 30 min).

In some embodiments, the pre-activation treatment is at a suitable temperature and a small amount of CO₂In an incubator according to (1). In some preferred embodiments, the pre-activation treatment is 5% CO at 37 ℃₂In an incubator.

It should be noted that the tetraploid blastocyst cannot normally develop, but can form a placenta, and can be used for verifying whether the stem cell has totipotency. The tetraploid blastocyst is a tetraploid embryo generated by externally stimulating two diploid embryos of 2 cells.

In some embodiments, the tetraploid blastocyst is obtained by an electrofusion or chemical fusion process.

In some embodiments, the tetraploid blastocyst is obtained by the following method: obtaining a mammalian 2-cell stage embryo, placing the 2-cell stage embryo into an embryo fusion liquid, and shocking the 2-cell stage embryo with an electrofusion apparatus at a direct-current electric field strength of 1-3kV/cm (such as 2kV/cm) and a pulse time interval of 30-50 μ s (such as 40 μ s) to obtain the tetraploid blastocyst (such as obtaining the tetraploid blastocyst with two sets of chromosome sets); optionally, the blastocysts are cultured (preferably at a suitable temperature and a small amount of CO) in a medium such as M16 medium₂More preferably, the culturing is carried out at 37 ℃ and 5% CO₂Performed in an incubator) for use.

In some embodiments, the mammal is a non-human mammal, e.g., the mammal is a mouse, sheep, cow, pig, or monkey.

In some embodiments, step 2) is performed by:

and (3) sorting haploid embryonic stem cells in G0-G1 stages by using a flow cytometric sorter. The nucleus of a haploid embryonic stem cell with a complete four-gene knockout was then injected into a pre-activated oocyte using a micromanipulator. After injection, the reconstituted embryos are placed in a 10Mm concentration Srcl solution₂The calcium-free CZB medium was cultured for 5 hours, and after 5 hours the reconstituted embryos were placed into M16 for further culture. In some preferred embodiments, the culturing is at a suitable temperature and a small amount of CO₂In an incubator according to (1). In some more preferred embodiments, the culturing is at 37 ℃ with 5% CO₂In an incubator.

In some embodiments, step 3) is performed by:

and (3) putting the blastocyst obtained in the previous step into 5mg/ML streptokinase protease, putting the blastocyst into an environment at 37 ℃ for 3 minutes, and removing the zona pellucida. The zona pellucida-removed blastocysts were then placed in DMEM medium containing 10% fetal bovine serum and 20% whole anti-mouse serum, at a suitable temperature and with a small amount of CO₂An incubator (e.g., 37 ℃, 5% CO)₂Incubator) for 3 hours. The blastocysts were then washed with DMEM/fetal bovine serum (10%) culture and incubated in 100% mouse serum for 20 min. In some preferred embodiments, prior to tetraploid compensation, the embryo is gently blown, the TE cells are gently removed, and the isolated ICM cells are placed in HER/FBS (GIBCO) medium.

In some embodiments, step 4) is performed by:

the inner cell mass cells obtained in the previous step are selected and injected into the quadruple blastula obtained by the previous method by using a micromanipulator, and about 15 inner cell mass cells are injected into each blastula.

In some embodiments, step 5) is performed by:

the blastocyst obtained by the aforementioned method is transplanted into the uterus of a pseudopregnant mouse for development, and then the mouse is dissected to obtain a fetus.

In some embodiments, step 6) is performed by:

and (3) soaking the obtained fetus in alcohol for 2h to sterilize surgical scissors and forceps, and removing the head and various organs of the fetus. The remaining embryos are minced and the minced tissue is digested in 2mL of 0.25% trypsin with 1mM EDTA in an incubator at a suitable temperature (e.g., 37 ℃) for 10 minutes. Diluting with DMEM liquid containing 10% fetal calf serum and penicillin/streptomycin to stop digestion reaction, and gently and repeatedly sucking for more than 20 times. Diluting the cell suspension with fresh culture medium, spreading on a 100mm culture dish, shaking, and adding small amount of CO at appropriate temperature₂An incubator (e.g., 37 ℃, 5% CO)₂Incubator) for two days, obtaining the somatic cell (such as fetal fibroblast) with single allele knockout of the target gene. In some preferredIn embodiments of (3), the somatic cells (e.g., fetal fibroblasts) are all first generation cells.

In a fifth aspect of the invention, the invention provides the use of the somatic cells described above or obtained by the methods described above for nuclear transfer.

In a sixth aspect of the invention, there is provided a method of nuclear transfer of somatic cells, which utilizes donor somatic cells for nuclear transfer, wherein the donor somatic cells are as described above.

In some embodiments, the somatic cell nuclear transfer method comprises:

a-1) injecting 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), and embryo fusion is performed to construct a third reconstructed embryo,

b-1) activating the third reconstructed embryo.

In some embodiments, the somatic cell nuclear transfer method comprises:

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), a fourth reconstructed embryo is constructed,

b-2) activating the fourth reconstructed embryo.

In some embodiments, step a-1) is performed by:

removing nucleus of target recipient cell (such as oocyte) by micromanipulator, putting the donor somatic cell (such as fetal fibroblast) into inactivated Sendai virus for several seconds, taking out, injecting the donor somatic cell with Sendai virus into the intercellular space (such as egg space) of enucleated target recipient cell (such as oocyte) by Piezo, and putting embryo into proper temperature and small amount of CO₂An incubator (e.g., 37 ℃, 5% CO)₂Incubator) until embryos fuse.

In some embodiments, step B-1) is performed by:

the fused embryos are transferred to a culture medium (e.g., M16 medium) for short culture, activated for 5-6h in calcium-free CZB medium containing 5mg/mL cytochalasin B and 10mM strontium chloride, and finally cultured in a culture medium (e.g., M16 medium).

In some embodiments, the culturing is at a suitable temperature and a small amount of CO in step A-1) or step B-1)₂An incubator (e.g., 37 ℃, 5% CO)₂Incubator).

In some embodiments, the oocyte is obtained by:

female mice of the appropriate age were selected, injected with Pregnant Mare Serotonin (PMSG), and injected with Human Chorionic Gonadotropin (HCG) within 13-17 hours after injection. Ovum pickup was performed 13-15h after HCG injection. The collected oocytes are washed with Hepes-CZB, cultured in a culture medium (e.g., M16 culture medium), and placed at a suitable temperature and a small amount of CO₂An incubator (e.g., 37 ℃, 5% CO)₂Incubator) for use. Granulosa cells were removed by hyaluronidase digestion and oocytes were kept in CZB medium containing 3mg/mL BSA for a short time before micromanipulation.

In a seventh aspect of the invention, the invention provides the use of the aforementioned modified haploid embryonic stem cell or the aforementioned somatic cell in the manufacture of a medicament for the treatment of a disease associated with somatic cell nuclear transfer.

In some embodiments, the disease associated with somatic cell nuclear transfer is fetal macrosomia syndrome.

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

In some embodiments, the disease associated with somatic cell nuclear transfer is overweight newborn fetus in a gestational diabetic mammal.

In some embodiments, the fetal large syndrome is mammalian (preferably non-human mammalian) fetal large syndrome.

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

In an eighth aspect of the present invention, there is provided a method of treating a disease associated with somatic cell nuclear transfer, comprising: nuclear transfer was performed using donor somatic cells, wherein the donor somatic cells were as described above.

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

In some embodiments, the method of treating a disease associated with somatic cell nuclear transfer comprises:

b-1) activating the third reconstructed embryo.

b-2) activating the fourth reconstructed embryo.

In a ninth aspect of the invention, the invention provides an animal wherein the animal has been single-allele knocked out for a gene selected from the group consisting 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, and any combination thereof.

In some embodiments, additional H19 and IG are single-allele knockouts in the animal, and optionally, additional Rasgrf1 is single-allele knockouts.

In some embodiments, additional H19, IG, and Rasgrf1 are single-allele knockouts in the animal.

In some embodiments, the animal is single allele knocked out of a gene selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, in the animal, Sfmbt2 is single allele knockout, and optionally, a gene selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof.

In some embodiments, in the animal, a gene or combination of genes selected from the group consisting of:

only Sfmbt2 is knocked out,

alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 were simultaneously knocked out.

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

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

In a tenth aspect of the invention, the invention provides a method of making the aforementioned animal comprising:

culturing the activated third or fourth reconstructed embryo obtained by the aforementioned method;

and (3) when the third reconstructed embryo or the fourth reconstructed embryo develops to a proper stage (such as 2 cells, or 4 cells, or 8 cells, or more cells), transferring the third reconstructed embryo or the fourth reconstructed embryo to an animal oviduct or uterus, and obtaining the animal at term.

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

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

Has the advantages that:

single allele knock-out on fibroblasts of imprinted genes of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, tl 3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (one or more of four as Sfmbt2, Jade1, Gab1 and Smoc1) H3K27me3 makes the expression pattern of these genes imprinted to be normal. Meanwhile, the cloning efficiency of the fibroblast can be increased to 14% by using the fibroblast for cloning, and the cloning efficiency of the fibroblast donor cell of a wild control group is 0. In addition, the cloned animal obtained by the method has corrected large placenta-large fetus phenomenon. Among the four genes, Sfmbt2, Jade1, Gab1 and Smoc1 are single genes with the most remarkable improvement of cloning efficiency in a single gene knockout strategy of Sfmbt 2.

Drawings

Figure 1 shows the loss of H3K27me3 imprinting in cloned placenta, wherein:

a represents a histogram of the relative gene expression levels of the autosomal imprinted gene in the E10.5 cloned placenta. Shows 50 imprinted genes detected in cloned placenta (FPKM > 1). The expression level of the placental imprinting gene of the in vitro fertilized fetus was set to 1. The gene framed by the box was named H3K27me3 imprinted gene. The horizontal dashed line indicates the overexpressed imprinted gene in the cloned placenta (fold change >2),

b represents a histogram of the relative expression levels of the X chromosome-linked imprinted genes in the E10.5 cloned placenta. The expression level of the placental genes from in vitro fertilization was 1. The horizontal dashed line indicates the overexpressed imprinted gene in the cloned placenta (fold change >2),

c represents a histogram showing the H3K27me3 imprinted gene paternal allele expression ratio in the placenta in vitro fertilized and cloned identified by SNP. The gene names not boxed and boxed represent the classical and H3K27me3 imprinted genes respectively,

d represents the expression ratio of the parental allele of the H3K27me3 imprinted gene in the placenta of the in vitro fertilization and clone E19.5 by RT-PCR and Sanger sequencing detection by taking SNP as a marker,

e represents a protocol based on 4H 3K27me3 imprinted gene knockouts and MII oocyte injections of haeSCs3KO,

f indicates the wild type E9.5 embryo and placenta on the left. On the right, blocked 4H 3K27me3 imprinted gene knockouts prepared based on halESCs 3KO E9.5 fetuses and placentas. White triangles mark the placenta. The scale bar is 0.5 mm;

FIG. 2 shows the preparation and cloning of somatic cells of the H3K27me3 imprinted gene with 4 gene single-allele knockouts, wherein:

a shows the protocol for the preparation of Δ 4-MEFs3KO cells and their use as nuclear transfer donor cells for cloning. Δ 4-embryos3KO was injected into oocytes, their inner cell mass was separated when they developed into blastocysts, and ICM was injected into wild-type tetraploid blastocysts and then transplanted. Then, MEFs were prepared from E13.5. DELTA.4-embryos 3KO fetuses, and these MEFs were used to obtain. DELTA.4-NT-mic 3KO for donor cell clones,

b shows the immunofluorescence results of H3K27me3 imprinted genes of IVF, WT-NT and Δ 4-NT blastocysts. The red signal is H3K27me3 imprinted gene staining. The green signal is CDX2 staining and the blue signal is DAPI staining of DNA. The scale is 15 μm in size,

c shows a comparison of immunofluorescence of the H3K27me3 imprinted gene in IVF, WT-NT and Δ 4-NT blastocyst TE cells. Each dot represents the protein to DAPI intensity ratio for a single TE cell. The solid line represents the mean and standard deviation. "x" indicates that p is less than 0.0001, ns indicates that the difference is not significant, as measured by t-test;

FIG. 3 shows the development results of somatic cloned embryos carrying a single-allele knock-out of four H3K27me3 imprinted genes, wherein:

a represents the statistics of implantation rates using TSA treatment versus untreated wild-type MEF, Δ 4-MEFs and Δ 4-TTFs,

b shows photographs of Δ 4-NT-mie 3KO and placenta, which have just been dissected out in one experiment. The scale bar is 10mm, and the scale bar is,

c represents weight comparison of expired wild type cloned mice and Δ 4-NT-mic 3KO, in vitro fertilized pups as controls,

d represents expired wild type cloned mice,. DELTA.4-NT-mic 3KO placental weight comparison, in vitro fertilized pup placental weight control,

e shows a comparison of the diameters of the wild-type cloned mouse placenta and the. DELTA.4-NT-mica 3KO placenta. Placenta from in vitro fertilization served as a control group. "represents p less than 0.05," "represents p less than 0.01," "represents p less than 0,001," "represents p less than 0.0001, ns represents no significant difference, t-test analysis,

f is a placental section map, the upper HE staining map and the lower immunohistochemistry map for the Lamin. alpha.1 antibody. The yellow line of the upper panel and the red line of the lower panel mark the fetal vessels in the labyrinth layer, respectively. The scale bar is 1mm, and the scale bar is,

g represents the histogram of the proportion of fetal vessels in the labyrinth layer. ". indicates that p is less than 0.0001, analyzed by t-test,

h represents the section of the placenta of IVF, WT-NT and delta 4-NT-mic 3KO, the area marked with stars at the upper side is the vascular area, and the area marked with stars at the lower side is the cell-free area in the placenta. The scale bar is 0.15 mm. "" represents p less than 0.05, "" represents p less than 0.01, "" represents p less than 0.001, "". represents p less than 0.0001, ns represents no significant difference, as analyzed by t-test,

i represents a histogram of the proportion of cell-free regions in the labyrinth layer,

j represents a histogram of the proportion of fetal blood vessels in the labyrinth layer;

figure 4 shows that correcting a single H3K27me3 imprinted gene can improve SCNT embryo development to varying degrees, where:

a represents Delta 4-NT-mic 3KO and its progeny. The offspring with asterisks are dead newborn mice, and the mice inherit IG-DMR knockout from the female parent,

b represents corrected litter size of Δ 4-NT-mie 3KO, comparable to the WT group,

c represents the Δ 4-NT-mie 3KO progeny, Sfmbt2 Δ/+ mouse and WT mouse brain transcriptome comparisons. n is 2. "" represents p less than 0.05, "" represents p less than 0.01, "" represents p less than 0.001, "". represents p less than 0.0001, ns represents no significant difference, as analyzed by t-test,

d denotes WT-TTFs, Sfmbt2^Δ/+-TTFs，Jade1^Δ/+-TTFs，Gab1^Δ/+-TTFs，Smoc1^Δ/+-TTFs and Δ 3-TTFs implantation rates are compared,

e denotes Sfmbt2^Δ/+-a photograph of NT mice,

f is a placental section map, the upper HE staining map and the lower immunohistochemistry map for the lamin α 1 antibody. The yellow line of the upper panel, and the red line of the lower panel mark the fetal vessels in the labyrinth layer. The scanned images of each sample are combined into one image by scaling. The scale bar is 1mm,

g represents the histogram of the proportion of fetal vessels in the labyrinth layer. The data presentation in all figures is: mean ± standard deviation. "+" indicates that p is less than 0.01, "+" indicates that p is less than 0.001, ns indicates no significant difference, analyzed by t-test,

h represents a schematic diagram of a placenta-specific imprinting mode of an H3K27me3 imprinted gene in a fertilized embryo and a rescue mode diagram of SCNT cub low rate and placenta/offspring defects by cloning H3K27me3 heterogeneously single-allele knocked-out somatic cells.

In all of the above graphs, data are presented as mean ± variance. T-test values p <0.05, p <0.01, p <0.001, p < 0.0001;

FIG. 5 shows an analysis of the expression of the H3K27me3 imprinted gene in cloned embryos, in relation to FIG. 1, where:

a represents a histogram of the relative gene expression levels of autosomal imprinted genes in the E10.5 cloned fetuses. 59 imprinted genes (FPKM >1) detected in cloned embryos were more reliable. The gene expression level of the in vitro fertilized embryo was set to 1. The gene name enclosed by a box is the H3K27me3 imprinted gene. The horizontal dashed line indicates that the imprinted gene was overexpressed in cloned embryos (fold change >2),

b indicates the relative expression level of the X-chromosomal imprinted gene in the E10.5 cloned fetuses. The gene expression level in the in vitro sperm-receiving embryos was set to 1.

C represents the ratio of H3K27me3 imprinted gene paternal allele expression (paternal to maternal) in vitro fertilized and cloned embryos identified by SNP. The gene names not framed by the boxes and the gene names framed by the boxes are the classical imprinted gene and the H3K27me3 imprinted gene, respectively,

d, top knockout scheme for exon 3 of Jade 1. The sgRNA sequence for knockout. Next, as a result of Sanger sequencing of genome and cDNA, it was confirmed that a 320bp DNA deletion of exon 3 of Jade1 (left) and a frame shift mutation of Jade1 mRNA (right),

e, knockout scheme of Smoc1 exon 4. The sgRNA sequence for knockout. In addition, as a result of Sanger sequencing of genome and cDNA, 639bp DNA deletion of exon 4 of Smoc1 (left) and frameshift mutation of Smoc1 mRNA (right) were confirmed,

in F, the upper exon 3 knockout scheme of Gab 1. The sgRNA sequence for knockout. Next, as a result of Sanger sequencing of genome and cDNA, 773bp DNA deletion of exon 3 of Gab1 (left) and frame shift mutation of Gab1 mRNA (right) were confirmed,

g, knockout of Sfmbt2 exons 16-17. The sgRNA sequence for deletion. Next, as a result of Sanger sequencing of genome and cDNA, 3290bp DNA deletion of exons 16 to 17 of Sfmbt2 (left) and frameshift mutation of Sfmbt2 mRNA (right) were confirmed,

h represents the result of immunoblotting detection of H3K27me3 imprinted gene in WT haeSCs and delta 4-haeSCs3KO,

i represents a gene modification diagram of a haploid embryonic stem cell replacing sperm;

fig. 6 shows gene expression and survival curves characterizing Δ 4-NT pups, correlated with fig. 2, wherein:

a represents a Western blot showing the expression of the H3K27me3 imprinted gene in expired WT-NT, WT-IVF and Δ 4-NT-mic 3KO placentas,

b shows brain transcriptome comparison of Δ 4-NT-mic 3KO with WT mice. n is equal to 2, and n is equal to 2,

c represents the cloned mice of the delta 4-TTFs3KO and the placenta thereof born in one experiment,

d represents the survival curves of WT and Δ 4-MEFs/TTFs cloned mice;

figure 7 shows the correlation of Δ 4 and Xist deletions in mouse SCNT, in relation to figure 3, where:

in A, upper: knock-out protocol for Xist gene. In the middle, the sgRNA sequence used for the Xist knockout was used. Next, Sanger sequencing of the genome and cDNA showed 17301bp knock-out of the Xist gene.

B represents delta 4-NT-mie^3KOAnd Δ Xist/Δ 4-NT-mica^3KOTranscriptome comparisons of brain. n is 2.

C represents delta 4-NTmica of E19.5^3KOAnd Δ Xist/Δ 4-NT-mica^3KORelative expression level histograms of X-chromosomally imprinted genes of placenta. Delta 4-NT-mie^3KOThe expression level of the placenta was set to 1. n is 2.

D represents the just cut-out Δ 4-TTFs^3KOPhotographs of cloned mice and placenta are taken,

e denotes the Southern blot hybridization results confirming Xist^Δ/YSingle allele knock-out of Xist in mice

In F, left panel: TTF^3KOEmbryo-transferred uteri were cloned. Right panel: xist dissected from a single uterus^Δ/YTTFs cloning of fetuses and implantation sites. The scale is 2 mm.

G represents the ratio of fetuses at different stages to all reconstructed fetuses. 2-cell stage embryos are considered E1.0; the dissected out implantation point is regarded as E5.0; a white-eye fetus is considered to be less than E10.5 in developmental stage; a black-eye fetus is considered to have a developmental stage greater than or equal to E10.5; fetuses with both black and white eyes are also considered to be less than E10.5 in developmental stage. Xist^Δ/Y-TTF, n ═ 5; WT-MEF and Δ Xist/. DELTA.4-MEF^3KO，n＝4；△4-MEF^3KOAnd Delta 4-TTF^3KOAnd n is 3. "indicates that p is less than 0.05""represents p less than 0.01," "×" represents p less than 0.001, "×" represents p less than 0.0001, ns represents no significant difference, analyzed by t-test;

figure 8 shows the contribution of the H3K27me3 imprinted gene alone to the development of the cloned placenta, in relation to figure 4, wherein:

a represents a single H3K27me3 imprinted gene single-allele knockout TTFs acquisition scheme,

b represents delta 4-NT-mie^3KOThe result of the genotype identification of the surviving offspring,

c indicates that the contribution of different imprinted genes to the clone is different,

d and E denote Sfmbt2^Δ/+-NT and Δ 3-NT fetal body weight and placental re-analysis,

f represents WT-NT, Sfmbt2^Δ/+-section images of the vascular zone (upper) and cell-free discontinuity zone (lower) of the placenta of NT and Δ 3-NT maturing mice. The regions marked with asterisks are the vascular zone and the cell-free discontinuous zone, respectively. The scale is 0.15mm and,

g represents a bar graph of the area ratio of the cell-free zone in the labyrinth layer,

h represents the area ratio histogram of the vascular zone in the labyrinth layer. "+" indicates that p is less than 0.05, "+" indicates that p is less than 0.01, "+" indicates that p is less than 0.001, "+" indicates that p is less than 0.0001, and ns indicates no significant difference, as measured by t-test.

Detailed Description

The technical solution of the present invention is clearly and completely described by the following specific examples. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Successful cloning by somatic cell nuclear transfer requires overcoming a significant epigenetic barrier. Although H3K27me 3-dependent imprinted genes may exhibit differential expression in embryonic epiblast and extraembryonic tissues at embryonic stage E6.5 days, genomic imprinting is not generally considered to be a barrier to nuclear transfer failure. The inventor reports a method for remarkably improving the nuclear transfer efficiency, and the method utilizes a haploid embryonic stem cell system to obtain donor somatic cells with single allele knock-out of one or more of 26H 3K27me 3-related imprinted genes for nuclear transfer. The results show that single-allele knockout of imprinted genes of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, tl 3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (one or more of four, such as Sfmbt2, Jade1, Gab1 and Smoc1) H3K27me3 makes the imprinted expression patterns of these genes tend to be normal. Meanwhile, the cloning efficiency of the fibroblast can be increased to 14% by using the fibroblast for cloning, and the cloning efficiency of the fibroblast donor cell of a wild control group is 0. In addition, the cloned animal obtained by the method has corrected large placenta-large fetus phenomenon. Among the four genes, Sfmbt2, Jade1, Gab1 and Smoc1 are single genes with the most remarkable improvement of cloning efficiency in a single gene knockout strategy of Sfmbt 2. The above results indicate that somatic cells lacking H3K27me3 imprinting are important barrier factors for preventing the development of somatic cell nuclear transfer embryos after implantation, and that the simultaneous modification of the expression pattern of these imprinted genes in donor cells can overcome these barriers of nuclear transfer.

The invention is further illustrated by the following examples.

The sequencing data are stored in the genome sequence file of Beijing genome research institute of Chinese academy of sciences, and the accession number is CRA002383(http:// gsa. big. ac. cn). A list of software for data analysis and processing can be found in the "key resources table".

All mouse experiments were performed according to the animal research instruction published by the animal research institute of the Chinese academy of sciences. All mice were housed in an animal facility at the academy of sciences of china. B6D2F1(C57BL/6 xDBA/2), C57BL/6 and CD-1 background mice were purchased from Wintolite, Beijing. PWK/PhJ (stock No. 003715) mice and C57BL/6-Tg (CAGEGFP)1Osb/J (stock No. 003291) were purchased from Jackson laboratories. Male C57BL/6 mice were used to mate with Δ 4-NT mice to obtain offspring carrying an isolated deletion of the non-classically imprinted gene. Female C57BL/6-Tg (CAGEGFP)1Osb/J XPWBK/PhJ mice were used to provide cumulus cells for nuclear transfer. Female B6D2F1 mice (C57BL/6 xDBA/2) were used to provide somatic donor cells and oocytes, and CD-1 background mice were used to provide fertilized embryos and pseudopregnant females.

The sources of key reagents involved in the following examples are shown in table 1 below.

Table 1: sources of Key Agents

In addition, the specific steps of some of the experimental procedures involved in the following examples are as follows:

1. collection of oocytes

Female mice of the appropriate age were selected, injected with Pregnant Mare Serotonin (PMSG), and injected with Human Chorionic Gonadotropin (HCG) within 13-17 hours after injection. Ovum pickup was performed 13-15h after HCG injection. The collected oocytes were washed with Hepes-CZB, then cultured in M16(Sigma) medium, and placed in a 37 ℃ 5% CO2 incubator until use. Granulosa cells were removed by hyaluronidase digestion and oocytes were kept in CZB medium containing 3mg/mL BSA for a short time before micromanipulation.

2. CRISPR-CAS9 gene editing

According to table 2 the inventors constructed a pair of sgRNAs for each of the four genes of each patent of the present invention, these sgRNAs targeted the universal exon regions of all transcripts of the respective genes, while Xist was targeted to exons 1-6. sgRNAs and Cas9 were subsequently transfected into 10 using a neo (Invitrogen) electrotransfer instrument⁶Two days later, GFP-positive haESCs were sorted by flow cytometry and seeded at low density onto feeder cells,after 7 days, individual colonies were picked for PCR detection. Unlike the method of the inventors to obtain MEFs with quadruplicate non-classical imprinted gene deletions, the inventors directly transfected Δ 4-MEFs via Cas9 and xgrna-knockout expression plasmids^3KOProduction of Δ Xist/Δ 4-MEFs on cell lines^3KO. Subsequently transfected A4-MEFs^3KOThe cell nucleus of (a) is injected into an enucleated oocyte for cloning, and the reconstructed embryo is transferred into a pseudopregnant recipient. E13.5 embryos were taken to obtain a new fibroblast line MEFs, embryos with Xist single allele knock-outs were screened by PCR and confirmed by sanger sequencing. Xist was obtained by injecting Cas9 and Xist-knockout sgRNA expression plasmids into one-cell stage embryos of C57BL6 mice^Δ/YA mouse. The genotype of the mice was verified by PCR and sanger sequencing. Xist-knockout female mice were mated with C57BL6 male mice to produce Xist^Δ/YA mouse. Xist^Δ/YThe genotype of the mice was confirmed by Southern hybridization.

3. Intracytoplasmic injection of haploid stem cells (Delta 4-haeSCs)^3KO)

(1) The superovulation method is as in method 1, and MII stage oocytes of the B6D2F1 strain are obtained and kept in an incubator for use.

(2) By flow sorting of G0 or G1 stage Δ 4-haESCs^3KOThe cells serve as donors. Prior to microinjection, oocytes were placed in a medium containing 10mM SrCl₂The culture solution of CZB was cultured for 30 min. Followed by the addition of the Delta 4-haeSCs^3KOThe cells are injected into oocytes separately to construct reconstituted embryos.

(3) Reconstituted embryos in the presence of 10mM SrCl₂Activated for 5h in calcium-free CZB medium. Fully activated embryos were washed twice in M16 and finally cultured in M16 medium, incubated at 37 ℃ CO₂An incubator.

(4) The next day, 2-cell stage reconstituted embryos are either transplanted into pseudopregnant CD-1 dams or transferred to KSOM medium for culture to blastocysts.

4. Immunofluorescence of mouse embryo

All blastocysts were treated with 5mg/mL pronase (Sigma) at 37 ℃ and 5% CO2 for 5-10 min to remove zona pellucida, transferred to M16 for washing, and subsequently fixed with 4% Paraformaldehyde (PFA) for 30 min. After washing with PBS, the embryos were transferred to a medium containing 200. mu.l of a membrane permeabilizing solution containing 0.1% Triton X-100 and permeabilized for 30 min. Blocking for 1h in 1% BSA blocking solution containing 0.1% Tween-20 and 0.01% TritonX-100. The blocked embryos were transferred to primary antibody (CDX2and GAB1/JADE1/SMOC1) at 4 ℃ overnight. PBS was washed 3 times. And co-incubating with secondary antibody at room temperature for 1 h. The secondary antibodies used were Alexa 488donkey anti-mouse and Cyanine3 goat anti-rabbitt. Cell nuclei were stained, DAPI, 5min at room temperature. To compare the extraembryonic protein levels of the H3K27me 3-dependent imprinted gene, relative fluorescence intensities of GAB1, JADE1 and SMOC1 in trophoblast cells from IVF, WT-NT, and Δ 4-NT blastocysts were analyzed, and TE cells positive for CDX2 signal were selected for analysis. The ratio of GAB1/JADE1/SMOC1 protein to DAPI signal was recorded and analyzed in each TE cell (IMARIS 3D/4D Visualization & Analysis Software).

5. Tetraploid compensation

Transfer the Δ 4-3KO embryos to preheated pronase solution with a mouth pipette, remove the zona pellucida for 3min at 37 ℃. The blastocysts without zona pellucida were incubated in DMEM containing 10% fetal bovine serum and 20% whole anti-mouse serum for 3h at 37 ℃. Blastocysts were washed with DMEM/FBS (10%) medium and incubated in 100% mouse serum for 20 min. Prior to tetraploid compensation, embryos are gently blown, TE cells are gently removed, and the isolated ICM cells are placed in HER/fbs (gibco) medium.

Preparation of tetraploid compensated tetraploid blastocysts was obtained as previously described (Zhao et al, 2009.) briefly, 2 cell embryos were taken from the oviducts of CD-1 dams and electrofused to produce tetraploid embryos. 10 to 15 ICM cells were then injected into each blastocyst and transplanted into the uterus of CD-1 pseudopregnant females.

6. Donor cell preparation for somatic cell nuclear transfer

Fetal fibroblasts (MEFs) were derived from E13.5 Δ 4-3KO tetraploid offset embryos, E13.5B 6D2F1 embryos and E13.5 embryos from intracytoplasmic injection of haeSCs3 KO. Soaking sterilized surgical scissors and forceps in alcohol for 2 hr to remove fetal head and organs. The remaining embryos were minced, and the minced tissue was digested in 2ml of 0.25% trypsin containing 1mM EDTA in an incubator at 37 ℃ for 10 minutes. Diluting with DMEM liquid containing 10% fetal calf serum and penicillin/streptomycin to stop digestion reaction, and gently and repeatedly sucking for more than 20 times. The cell suspension was diluted with fresh medium, plated on a 100mm petri dish, shaken well, and cultured at 37 ℃ in a 5% CO2 incubator for two days. Cells were cryopreserved (passage 0). In all experiments, MEFs used first generation cells.

Tail tip fibroblasts (TFFs) were obtained from the tail tips of 6-week-old Δ 4-NT-3KO mice, B6D2F1 mice, and mice generated by intracytoplasmic injection of haeSCs3KO cells. The excised tail tip tissue was placed on the bottom of a dish pre-coated with fibronectin and dried at 25 ℃ to 27 ℃ for 10 min. Thereafter, DMEM containing 10% fetal bovine serum was gently added to avoid the tissue from falling off the disc bottom. The dishes were incubated at 37 ℃ until fibroblasts divide and pool at the bottom of the dish (passage 0). TTFs were used in all experiments with cells before passage 3.

Cumulus cells were obtained superimposingly by injection of 7.5IU of PMSG and 7.5IU of hCG from 8-week-old female mice (B6/PWK). After 15-17h, cumulus cells were collected by digesting oocytes with preheated 300IU/ml hyaluronidase. Prior to micromanipulation, the oocytes were kept in 3mg/mLBSA CZB medium, covered with petroleum, and placed in a 37 ℃ 5% CO2 incubator. Cumulus cells were washed twice with HEPES-CZB medium and kept at 4 ℃ until needed.

7. Somatic cell nuclear transfer and embryo culture

SCNT uses the "one-step method" reported by Zhou et al (2003). Granulosa cell cloning is the injection of granulosa cells into enucleated oocytes using Piezo; and the fiber clone is formed by fusing enucleated oocyte and fibroblast by using inactivated Sendai virus. Transferring the manipulated embryo into M16 culture solution for short-term culture, and activating in activating solution containing 5mg/ml cytochalasin B and 10mM strontium chloride for 5-6 h. Then cultured in M16 plus TSA for 4h, and finally cultured in M16 culture solution.

8. Embryo transfer

2-cloning embryo in cell stage, transplanting the embryo into the oviduct bulge part of the E0.5 pseudopregnant CD-1 female mouse; tetraploid blastocyst transfer is the transfer of embryos into the uterus of E2.5 pseudopregnant CD-1 dams.

9. Immunohistochemical staining and histological analysis

The placenta produced at term was fixed in 4% paraformaldehyde, then paraffin-embedded and serially sectioned at a thickness of 4 mm, followed by HE staining. For immunohistochemical staining, sections were washed in PBS for 5 minutes, then incubated with blocking buffer (PBS containing 1% BSA and 0.1% Tween-20) for 20 minutes at room temperature, followed by incubation with primary antibody for 1 h. Thereafter, the sections were washed three times in PBS containing 0.1% Tween-20 and incubated with secondary antibodies for 1 h. Photographs were taken with a panoramic tissue cell analyzer (Leica Aperio VESA 8). Placental physiological parameters, including trophoblast area, decellularized disruptions and blood vessels, were analyzed using Im-ageScope (v12.0.1.5027) software.

10. RNA extraction and RT-PCR

The placenta of the fetus is digested into single cells, and flow sorting is carried out to sort out green fluorescent protein positive cells. RNA extraction was performed on GFP positive cells using the PureLinkTM RNA Mini Kit. Reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme), which was previously cleared using a gDNA scavenger provided in the cassette. PCR fragments containing PWK and C57 specific SNP sites were amplified using primers designed by PrimerPremier 5. The RT-PCR products were ligated using pClone007 Blunt Vector Kit (TsingKe). After transformation, the cells were incubated at 37 ℃ for 12h, after which the single clones were picked for sequencing analysis.

11. Preparation of RNA-Seq library and data analysis

The placenta is cut up and digested by 0.25% trypsin at 37 ℃, in the digestion process, the placenta is blown and sucked by a gun head for several times, so that the digestion is more comprehensive, and the digestion is stopped after 10-15 min. And (4) carrying out flow sorting on GFP positive placental cells, and carrying out RNA sequencing analysis after collection.

For transcriptome analysis of mice, WT mice were collected,. DELTA.4-NT-3 KO and RNA-Seq was performed from brain tissue of the mice. Total RNA was extracted from the fetus with TRIzol, and then reverse transcription polymerase reaction was performed with 1. mu.g of purified RNA each time.

RNA-seq database construction and data analysis: RNA purification of the PolyA tail was performed for 2 rounds of each sample. The 150bp paired end sequencing was performed using an Illumina HiSeq 4000 sequencer. RNA-seq data were analyzed using HISAT2(version 2.1.0) and Cufflinks (version 2.2.1). And (3) data statistics: the statistics and analysis of the partial data were performed using GraphPad software. One-way anova was used.

12. Protein immunoblotting (Western)

To obtain fresh placental cells developed from the fetus, the inventors minced the placenta and digested with 0.25% trypsin at 37 ℃, 5% CO2 for 10-15 minutes. And the minced tissue is blown and sucked for a plurality of times during the digestion process, and then GFP positive placental cells are sorted and collected by a flow cytometer. Placental cells or haESCs were dissolved in Pierce IP Lysis Buffer, placed on ice, and added with protease inhibitor and sodium orthovanadate every 30 min. Followed by centrifugation at 12000rpm for 10min in a4 ℃ centrifuge, the supernatant was collected and mixed with 10mL of sample Buffer (1.25mL of 0.5M pH6.8Tris-HCl,2.5mL of glycerol, 2mL of 10% SDS,200mL of 0.5% bromophenol blue, 3.55mL of H2O, and 0.5mL of. beta. -mercaptoethanol) and boiled in boiling water for 5 min. The samples were separated by SDS-PAGE and were 100V, 1h in 10mL of 5% compression gel (5.7mL ddH 2O,2.5mL 1.5M pH6.8Tris-HCl, 1.7mL 30% acylamide [ acyl: bis acyl 29:1],100mL 10% SDS,50mL 10% ammonium persulfate, and 10mL TEMED) and 10mL separation gel (4.1mL ddH 2O,2.5mL 1.5M pH 8.8Tris-HCl,3.3mL 30% acylamide [ acyl: bisacryl 29:1],100mL 10% SDS,50mL 10% ammonium persulfate, and 5mL TEMED). Then, the mixture was electrophoresed at 200mA at 4 ℃ for 1 hour and transferred to nitrocellulose. Subsequently, the membranes were blocked for 1 hour at room temperature in TBST buffer (10mM Tris,150mM NaCl, 0.1% Tween20, pH 7.4) containing 3% BSA (Sigma).

Then incubated with primary antibody (diluted in TBST with 1% BSA) at 4 ℃ overnight. Signals were detected using ECL after three washes (10 minutes each) in TBST, 1 hour incubation with secondary antibody at room temperature, and three washes (10 minutes each).

13、Southern blot

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

In addition, the sequences involved in the following examples are shown in table 2 below.

Table 2: primer name and sequence

Example 1 the occurrence of abnormal biallelic expression of the H3K27me3 imprinted gene in the E19.5 cloned placenta

In order to study the specific expression of the H3K27me3 imprinted gene, offspring of two inbred mice were used as study subjects in the present invention.

The experimental method comprises the following steps:

the female parent is a female mouse of C57BL/6-Tg (CAG-EGFP)1Osb/J strain, and the male parent is a male mouse of PWK/PhJ strain. Somatic cell nuclear transfer was performed using granulosa cells from crossed F1 generation mice, as shown in Experimental method 7 (i.e., somatic cell nuclear transfer and embryo culture), above. Embryos fertilized in vitro with C57(GFP +) and PWK sperm and oocytes were used as controls. These imprinted genes were not detected after E9.5. Thus, the inventors analyzed embryos for E10.5 SCNT and IVF. Fetal-derived placental cells are sorted using flow cytometric sorting techniques with green fluorescence, so that contamination of maternal cells does not occur. These sorted cells were then subjected to RNA-seq analysis.

The experimental results are as follows:

compared with the in vitro fertilization control, the expression levels of Sfmbt2, Smoc1 and Jade1 in 4H 3K27me3 imprinted genes of cloned placental cells were increased by more than 2-fold (fig. 1A). But only Sfmbt2 was overexpressed in the fetus (fig. 5A). Furthermore, Xist was also expressed 2-fold higher in the cloned placenta, while Xist expression was not too high in the cloned fetuses (fig. 1B and fig. 5B). Many classical imprinted genes, including H19, Meg3, and Cdkn1c, were also aberrantly expressed in cloned fetuses of E10.5 (fig. 1A and 5A).

Further, the inventors have conducted intensive studies on the specific expression of alleles by Single Nucleotide Polymorphism (SNP) analysis. Analysis showed that the expression pattern of the classical imprinted gene and the H3K27me3 imprinted gene remained unchanged in the cloned fetuses (fig. 5C and table 3). In contrast, in the cloned placenta, the allele-specific expression pattern of the 4H 3K27me3 imprinted genes was lost, whereas the typical imprinted genes were not (fig. 1C and table 3).

Further, the inventors analyzed the status of H3K27me3 imprinted gene in late placenta, cloned GFP positive cells in placenta by isolating E19.5 in vitro fertilization, performed reverse transcription PCR and Sanger sequencing analysis. The results showed that these 4H 3K27me3 imprinted genes still maintained a paternally biased expression pattern in the E19.5 in vitro fertilized placenta, whereas this pattern disappeared in the cloned placenta (fig. 1D). In addition, the H3K27me3 imprinted gene was imprinted missing in the cloned blastocyst. Thus, aberrant biallelic expression of atypical imprinted genes is maintained throughout the post-implantation development of cloned embryos.

Table 3: location of parental SNPs and enumeration of reads comprising SNPs

TABLE 3 continuation of the table

Example 24 single-allele knockout of H3K27me3 imprinted gene can obviously improve cloning efficiency

The experimental method comprises the following steps:

the invention discloses a method for improving cloning efficiency of a mouse, and a cell strain and a model mouse which can be used for improving cloning efficiency. The method comprises the following specific steps:

1. and (4) obtaining the oocyte. Female mice of the appropriate age were selected, injected with Pregnant Mare Serotonin (PMSG), and injected with Human Chorionic Gonadotropin (HCG) within 13-17 hours after injection. Ovum pickup was performed 13-15h after HCG injection. The collected oocytes were washed with Hepes-CZB, then cultured in M16(Sigma) medium, and placed in a 37 ℃ 5% CO2 incubator until use. Granulosa cells were removed by hyaluronidase digestion and oocytes were kept in CZB medium containing 3mg/mL BSA for a short time before micromanipulation.

2. Haploid embryonic stem cells which can replace sperms and are simultaneously knocked out by Sfmbt2, Smoc1, Gab1 and Jade1 genes are obtained. A pair of sgrnas was set for each of the four genes Sfmbt2, Smoc1, Gab1, Jade1 according to table 2, which sgrnas targeted the universal exon regions of all transcripts of the respective genes (fig. 5D-G). The inventors then electroporated plasmids carrying these sgrnas and Cas9 into H19, IG, and Rasgrf1 knockout (fig. 5I) haploid embryonic stem cells with green fluorescent protein expression using an electrotransfer instrument. The order of cells is 10^ 6. After the electrically transformed haploid embryonic stem cells are cultured on a haploid embryonic stem cell culture medium for two days, a flow cytometry sorter is utilized to sort out the haploid embryonic stem cells with green fluorescent protein, and then the PCR technology is utilized to identify the transfected cells, so that the haploid embryonic stem cells with the four completely knocked-out genes are identified. The primers were identified as in Table 2.

3. In vitro fertilization simulation was performed using the identified four gene knockout haploid embryonic stem cells as surrogate sperm. Using flow cytometryAnd (3) sorting haploid embryonic stem cells from G0 to G1 stages by a sorter. The oocytes that have been harvested are then pre-activated for 30 minutes in 10Mm concentration of calcium-free CZB culture medium containing Srcl 2. Haploid embryonic stem cells (delta 4-haeSCs) with four completely knocked-out genes^3KO) Is injected into the pre-activated oocyte using a micromanipulator. After the injection, the reconstructed embryo is put into a 10 Mm-concentration Srcl2 calcium-free CZB culture solution to be continuously cultured for 5 hours, and after 5 hours, the reconstructed embryo is put into M16 to be continuously cultured. The embryos were cultured in a 5% CO2 incubator at 37 ℃ for both pre-activation and culture. And (5) waiting for the embryo to develop into the blastocyst at E3.5 days.

4. And obtaining the tetraploid blastocyst capable of replacing the extraembryonic tissue. Obtaining a 2-cell-stage embryo of a mouse, putting the 2-cell embryo into a mouse embryo fusion liquid, shocking the embryo by using an electric fusion instrument under the conditions that the direct-current electric field intensity is 2kV/cm and the pulse time course is 40 mu s to obtain a tetraploid mouse reconstructed embryo with two sets of chromosome groups, and then putting the embryo into an M16 culture solution to be cultured for E3.5 days for later use. The embryos were all cultured in a 5% CO2 incubator at 37 ℃.

5. Obtaining the diploid inner cell mass cell with four genes knocked out. The inventors put the blastocyst obtained in step 3 into 5mg/ML streptokinase protease and put it in an environment of 37 ℃ for 3 minutes to remove the zona pellucida. The zona pellucida-removed blastocysts were then placed in DMEM medium containing 10% fetal bovine serum and 20% whole anti-mouse serum and incubated at 37 ℃ in a 5% CO2 incubator for 3 hours. The blastocysts were then washed with DMEM/fetal bovine serum (10%) culture and incubated in 100% mouse serum for 20 min. Prior to tetraploid compensation, embryos are gently blown, TE cells are gently removed, and the isolated ICM cells are placed in HER/fbs (gibco) medium.

6. Obtaining the fetal fibroblast donor cells with four gene knockout. And (4) selecting the inner cell mass cells obtained in the step (5), injecting the inner cell mass cells into the blastula obtained in the step (4) by using a micromanipulator, and injecting about 15 inner cell mass cells into each blastula. The blastocyst was then transplanted into the uterus of a 2.5 day pseudopregnant mouse, and the mouse was dissected as it was pregnant up to day E13.5 to obtain a fetus at embryonic stage day E13.5. The fetus was then soaked in alcohol for 2h with sterilized surgical scissors, forceps, and the fetal head and various organs were removed. The remaining embryos were minced, and the minced tissue was digested in 2ml of 0.25% trypsin containing 1mM EDTA in an incubator at 37 ℃ for 10 minutes. Diluting with DMEM liquid containing 10% fetal calf serum and penicillin/streptomycin to stop digestion reaction, and gently and repeatedly sucking for more than 20 times. Diluting the cell suspension with fresh culture medium, spreading on a culture dish of 100mm, shaking up, and culturing at 37 ℃ in a 5% CO2 incubator for two days to obtain four knockout fetal fibroblasts. Cells were cryopreserved (passage 0). In all experiments, first generation cells were used for fetal fibroblasts.

7. Nuclear transfer was performed using donor cells. The enucleated oocyte was enucleated using a micromanipulator, and then the fetal fibroblast obtained in step 6 was put into inactivated sendai virus for several seconds and then removed, and then the donor cell with sendai virus was injected into the egg space of the enucleated oocyte using Piezo, and then the embryo was put into a 37 ℃, 5% CO2 incubator until the embryo was fused. Then the fused embryo is transferred to M16 culture solution for short-term culture, and then is put into calcium-free CZB culture solution containing 5mg/ml cytochalasin B and 10mM strontium chloride for activation for 5-6h, and finally is cultured in M16 culture solution. The embryos were all cultured in a 5% CO2 incubator at 37 ℃. When the embryos developed to 2 cells, embryos were cloned at the 2-cell stage and the embryos were transferred into the oviduct puffs of E0.5 pseudopregnant CD-1 mice. Mice were waited for dissection due. By the time of the embryonic stage E19.5 days of the mice, 4 knockout expired mice were dissected from birth.

The process is shown in FIG. 2A.

Discussion of the experiments:

to solve the problem of loss of H3K27me3 imprinting in SCNT embryos, single-allele knockout of 4H 3K27me3 imprinting genes in somatic cells is required for recovery. Currently, allele-specific gene knock-out can be achieved by targeting DNA sites with SNPs on different alleles or using suboptimal, distance-dependent gene editing strategies, but it is difficult to practice to knock-out four single alleles simultaneously in somatic cells or embryonic stem cells. Meanwhile, since mice heterozygous for mutations at Sfmbt2 or Gab1 locus are paternally sterile, it is impossible to generate mice with single allele deletion in which four genes are knocked out simultaneously by mating mice heterozygous for different heterozygous mutations.

Therefore, the inventor uses the haploid embryonic stem cell (haESCs3KO) which is developed by the inventor's laboratory and is knocked out by three DMR of H19, IG and Rasgrf1 as a knocking-out platform, the haploid embryonic stem cell can maintain the paternal imprinting pattern during the process of passage and operation, and the haploid embryonic stem cell can be injected into an oocyte with the efficiency similar to that of a spherical sperm.

It is worth mentioning that mice produced by injection of oocytes of haESCs can also produce viable offspring by natural mating with wild-type males.

The experimental results are as follows:

through CRISPR-Cas9 gene knockout technology, the inventors made a frame shift mutated stem cell line (delta 4-haESCs3KO) of four genes Sfmbt2, Jade1, Smoc1 and Gab1 (FIG. 5D-H).

After obtaining the four H3K27me3 imprinted gene knockout haploid stem cell lines, the inventors obtained a reconstructed embryo (Δ 4-embryos3KO) by injecting Δ 4-haESCs3KO into the oocyte, transplanted it into a pseudopregnant female mouse, and observed its developmental competence (fig. 1E). Through dissection, the inventors found that Δ 4-embryos3KO can only develop to E9.5, and that its placental development is abnormal, indicating that the single-allele expression of the H3K27me3 imprinted gene plays a very critical role in the development of extra-embryonic tissues (fig. 1F and table 5).

To obtain a somatic cell nuclear transfer donor with a single allelic mutation of the H3K27me3 imprinted gene. The inventors isolated inner cell mass cells of the Δ 4-embryos3KO blastocysts by using CZB medium without calcium and magnesium, then injected these inner cell mass cells into quadruplicate blastocysts of wild-type ICR strain mouse embryo origin, and transplanted the embryos into surrogate mother's uterus after the procedure was completed (fig. 2A). It was found that tetraploid-compensated reconstituted embryos could develop to maturity (table 5). The phenomenon that the developmental capacity of the Δ 4-embryos3KO embryo is rescued by the tetraploid blastocyst further demonstrates that extraembryonic tissue defects limit its development. Most importantly, at E13.5, the inventors successfully generated fibroblasts (. DELTA.4-MEFs 3KO) using the rescued fetuses (FIG. 2A).

The inventors performed somatic cell nuclear transfer experiments with Δ 4-MEFs3KO as donor cells. Prior to this, the inventors' laboratory reported a special preimplantation embryo culture method: d-culture method. The two-cell late stage is cultured by using M16 culture solution before, and the two-cell late stage is cultured by using KSOM culture solution after, and the method can obviously improve the development efficiency of cloned blastocysts. However, the efficiency of blastocyst development was not improved by using D-culture method or not, when the cloned embryos using the Δ 4-MEFs3KO cells as donor cells (Δ 4-NT-embryos3KO) were compared with the efficiency of development of cloned embryos using wild-type MEFs. These results indicate that H3K27me3 imprinted gene knock-out did not affect the development of cloned embryos before implantation.

By immunofluorescent staining of blastocysts of IVF, WT-SCNT and Δ 4-NT-embryos3KO, the inventors found that in IVF embryos ICM, the signal intensities of Gab1, Jade1 and Smoc1 were similar to those in WT SCNT, but the intensities in TE were significantly reduced compared to those of WT clone TE (FIG. 2B). On the other hand, the signals in both ICM and TE were weaker than WT SCNT in the Δ 4-NT-embryos3KO blastocyst, indicating that the protein levels of Gab1, Jade1 and Smoc1 were effectively decreased following single allele knockdown in somatic cell nuclear transfer blastocysts (FIG. 2B). Relative fluorescence intensity analysis also showed that IVF and Δ 4-NT-embryos^3KOThe expression level of the H3K27me3 imprinted gene was lower in TE of blastocysts than in WT SCNT embryos (fig. 2C), indicating that the level of the H3K27me3 imprinted gene, which was overexpressed in TE of cloned embryos, was restored.

The inventors subsequently performed oviduct transplantation of Δ 4-NT-embryos3KO at the 2-cell embryonic stage. Compared with the control group, the implantation rate of the delta 4-NT-embryos3KO is greatly improved and is close to that of the in vitro fertilization group (FIG. 3A). Moreover, the maturity development efficiency of the delta 4-NT-embryos3KO is greatly improved to 8.5-14.2%, while the developmental stage efficiency of the wild type MEFs cloned embryos of the control group is 0%, and the maturity development efficiency of the wild type granulosa cell cloned embryos is only 1.1 +/-0.2%. (FIG. 3B and Table 4).

The body weight of the Δ 4-NT-embryos3KO newborn mice was also similar to that of the control group in vitro fertilized newborn mice, but lower than that of the cloned mice using wild-type granulosa cells (FIG. 3C). In addition, the weight of the mouse placenta cloned by delta 4-NT-mica 3KO was similar to that of the IVF group, but lower than that of the WT group (FIG. 3D). The diameter of the placenta was also such that the placenta diameter of Δ 4-NT-mic 3KO was similar to that of the in vitro fertilized mouse placenta and smaller than that of the WT clone group (FIG. 3E). Meanwhile, the protein immunoblotting result shows that the protein expression level of the H3K27me3 imprinted gene in the delta 4-NT-mica 3KO placenta is also reduced (FIG. 6A), and the fact that the overexpression of the H3K27me3 imprinted gene in the cloned placenta can be effectively recovered through knockout is proved. Furthermore, RNA-seq analysis showed that the brain tissues of the wild-type normal mice and the Δ 4-NT-mic 3KO mice had very similar transcriptomes and that the expression of the imprinted genes was similar (FIG. 6B). Eosin-hematoxylin staining and lamini a staining, a marker for fetal endothelium and its associated basement membrane, revealed that labyrinth fetal vessels were defective in the WT-MEF cloned placenta, and were largely rescued in the Δ 4-NT-mic 3KO placenta (fig. 3F-G). In addition, the cell-free discrete regions at the maternal-fetal interface, the hypo-placental vascular density phenotype, were also rescued (fig. 3H-J) (Georgiades et al, 2001).

In order to further study the influence of H3K27me3 imprinted gene knockout on somatic cell nuclear transplantation in adult cells, the inventors selected the tail tip of a 6-week-old Delta 4-NT-mie 3KO mouse to make a tail tip fibroblast (Delta 4-TTFs3 KO). Nuclear transfer with Δ 4-TTFs3KO as donor cells resulted in 4.8. + -. 1.0% of embryos that had developed to the end after transfer into surrogate mothers, whereas the birth efficiency of WT TTFs clones was only 0% (Table 4 and FIG. 6C). Although fibroblasts can be used for cloning, the cloning efficiency is very low, so that the cloning efficiency of the delta 4-MEFs3KO and the delta 4-TTFs3KO is still very considerable by the inventor.

The inventors tracked the survival of all Δ 4-MEFs/TTFs cloned mice, 23 Δ 4 cloned mice, 5 died at 21 days postnatally, 18 survived, and the overall survival was not different from that of wild type cloned mice (1 out of 5 died) (FIG. 6D). Taken together, these results indicate that correcting the aberrant biallelic expression of the four h3k27me3 imprinted genes can significantly increase the expiration rate of SCNTs, even for adult-derived fibroblasts.

Classical literature reports indicate that correcting abnormal expression of Xist can effectively improve the mature development efficiency of cloning. Therefore, the inventors transfected both Cas9 and Xist gene knockout sgRNA plasmids in Δ 4-MEFs3KO cells. Transfected Δ 4-MEFs3KO were used as donor cells for nuclear transfer, after which reconstituted embryos were transferred to surrogate mothers. The inventors obtained a fetus by dissecting the mother mouse at E13.5 and tested whether the fetus Xist had been knocked out by PCR and Sanger sequencing. The inventors used Xist single allele knock-out fetuses to make MEFs (Δ Xist/Δ 4-MEFs3KO) (FIG. 7A). However, the cloning efficiency was only 5.2. + -. 0.8% when Δ Xist/Δ 4-MEFs3KO was used as donor cells, and the efficiency of nuclear transfer was not further improved compared to that when Δ 4-MEFs3KO was used, indicating that knocking-out Xist did not work synergistically with the 4H 3K27me3 imprinted gene knock-out (Table 4 and FIGS. 7B-D).

Recovery of Xist levels did not improve pre-implantation development of SCNT embryos, and work by the Ogura group demonstrated that there was no statistical difference in blastocyst rate of Xist-siRNA injected cloned embryos compared to control siRNA injected embryos (Matoba et al, 2011). Meanwhile, when using testis support granulocyte or cumulus cell clone, the implantation rate of the somatic cell with single allele Xist deletion is no better than that of the WT cell. However, the cloning efficiency of both granulosa cells and testicular support cells is very high, and the implantation rates of both cells are high relative to fibroblasts, limiting the room for improvement of implantation rates by Xist knockouts. To better understand the potential synergy between Δ 4 and Δ Xist, the inventors used maternal Xist knockout mice to make Xist Δ/Y-TTFs as donor cells (fig. 7E). Similar to previous results, there was no improvement in cloned embryo blastocyst rates of Xist Δ/Y-TTFs compared to WT-MEF clones, whether or not D-culture was used (Table 6). Similar to the 4H 3K27me3 imprinted gene knockouts, Xist knockouts had no significant effect on preimplantation development of cloned embryos.

Subsequently, the inventors transplanted cloned embryos of 2-cell stage Xist Δ/Y-TTFs into pseudopregnant females. The dissected findings show that 65 implantation points of the transplanted 134 cloned embryos are obviously improved compared with that of only 5 implantation points of 240 embryos transplanted in a control group (table 4), and the results prove that the implantation rate of the cloned embryos can be improved by single-allele knocking out Xist in somatic cells.

Meanwhile, the inventors counted the survival rates of 5 different stages of the cloned embryo development in detail (2-cell stage/E1.0; implantation/E5.0; E10.5;. gtoreq.E 10.5; E19.5), and found that the 4H 3K27me3 imprinted genes and the Xist knockout contribution to the cloned embryo are very similar, and both the implantation rate and the post-implantation development rate of the cloned embryo can be improved (FIGS. 7F-G). However, unlike Δ Xist/Δ 4-MEFs3KO, the inventors did not produce mature mice in 134 Xist knockout cloned embryos (Table 4). These results provide a possible explanation for the lack of overlap between Δ 4 and Δ Xist during nuclear transfer development, since H3K27me 3-dependent imprinted genes, whether X-chromosome or autosomal, functionally overlap during clonal embryo development, and thus do not provide a good developmental efficiency overlap for correction.

On the other hand, the inventors' results also showed that the ability of the modified H3K27me3 imprinted gene to promote mature development was much stronger than Δ Xist when fibroblasts were used (table 4).

It has been reported in the classical literature that treatment of cloned embryos with the histone deacetylase inhibitor TSA significantly increases the efficiency of cloned embryo development. However, when the inventors treated embryos cloned with Δ 4-MEFs3KO with TSA, the development efficiency of the cloned embryos was not further improved, which indicates that there was no synergy between TSA treatment and the 4H 3K27me3 imprinted gene knockout (Table 4).

To identify the fertility of Δ 4-NT-mica 3KO mice, the inventors mated these cloned mice with wild-type males. Since all Δ 4-NT-mica 3KO mice are female, knocking out the H3K27me3 imprinted gene expressed by 4 parents does not affect the expression and function in offspring.

Δ 4-NT-mica 3KO mice were able to birth a viable fetus (FIG. 4A). Litter size was calculated for mice per litter by multiplying the number of surviving litters (litters not carrying H19 and IG-DMR knockouts) by 4, and the Δ 4-NT-mie 3KO litter size (n ═ 5) was 6.80 ± 2.71 comparable to 6.14 ± 1.80 for wild-type females compared to the litter size of wild-type female mice, suggesting a normal fertility (fig. 4B). In addition, RNA-seq analysis showed that the progeny of Δ 4-NT-mic 3KO had a normal transcriptome (FIG. 4C).

Example 3 fertilization-derived somatic cells with a single allele knock-out of the three/monogene H3K27me3 imprinted gene were able to improve placental development and to clone fetal development

The experimental method comprises the following steps:

the specific steps are basically the same as the experimental method of the example 2, and the differences are that: to obtain the donor cells required for this step, we mated Δ 4-NT-mic 3KO mice obtained in example 2 with wild-type male mice of C57BL6 to obtain isolated mice with single gene or multiple gene combinations, and used the fibroblasts of the mice for nuclear transplantation.

Discussion of the experiments:

to further investigate the effect of a single non-classical imprinted gene on cloning, the inventors used single-allele knockout TTF for cloning (transferred to as Sfmbt2 Δ/+ -TTFs, Jade1 Δ/+ -TTFs, Gab1 Δ/+ -TTFs, Smoc1 Δ/+ -TTFs) (fig. 8A). Progeny that did not carry the H19, IG and Rasgrf1 knockouts were used for cloning (fig. 8B).

The experimental results are as follows:

the inventors used these tail tip fibroblasts for nuclear transfer. The results show that the implantation rate of cloned embryos using Sfmbt2 Δ/+ -TTFs and Jade1 Δ/+ -TTFs was very significantly improved compared to that of the wild-type TTFs for nuclear transplantation (Table 4 and FIG. 4D). While there was only a slight increase in implantation rates of cloned embryos using Gab1 Δ/+ -TTFs and Smoc1 Δ/+ -TTFs (FIG. 4D), this difference was most likely due to the different genetic backgrounds of wild-type mice and Gab1 Δ/+/Smoc1 mice, rather than the ineffectiveness of imprinted gene knock-outs. The development efficiency was significantly improved with Sfmbt2 Δ/+ -TTFs clones that gave 2.4 ± 2.3% efficiency in the mature cloned mice compared to 0% maturation efficiency using wild-type TTFs clones (fig. 4E and table 4). Due to the significant effects of Sfmbt2 Δ/+ -TTFs and Jade1 Δ/+ -TTFs on post-implantation development (fig. 4D), the inventors examined the nuclear transfer efficiency of 2 gene single-allele knockouts of Sfmbt 2and Jade1, TTFs (Δ 2-TTFs) (fig. 8B). 73 embryos were transferred, no foetus was born, the implantation rate was 43.8%, similar to Sfmbt2 Δ/+ -TTFs (44.6%) and Jade1 Δ/+ -TTFs (46.2%) (Table 4). The inventors subsequently examined 3 single-allele knockouts of TTF (Sfmbt 2. delta./+, Jade 1. delta./+, and Gab 1. delta./+ [. delta. 3-TTFs ]), with a implantation rate of 53.4% for the.DELTA.3-TTFs, which was significantly higher than that of the wild-type cloned embryos (FIG. 4D and Table 4). In addition, the cloned Δ 3-TTFs had an efficiency of 4.6. + -. 0.6% for its embryos, similar to that of Δ 4-TTFs3KO (4.8. + -. 1.0%) (Table 4 and FIG. 8C). The body weights of Sfmbt2 Δ/+ -NT-mie (1.26. + -. 0.06g) and Δ 3-NT-mie (1.27. + -. 0.08g) were significantly reduced compared to the body weight of WT cloned mice (1.94. + -. 0.11g) (FIG. 8D). The placental weight of Δ 3-ntmica (0.15 ± 0.02g) was also significantly lower than WT cloned placenta (fig. 8E). Analysis of the placenta also showed a significant improvement in the proportion of labyrinthine fetal vessels in Sfmbt2 Δ/+ -NT-mica and Δ 3-NT-mica placentas (FIGS. 4F-G), but the defect of too low a placental vascular density and too high a proportion of cell-free discontinuity areas only improved in the Δ 3-NT-mica placentas, while these did not improve in the Sfmbt2 Δ/+ -NT-mica placentas (FIGS. 8F-H). Since genomic imprinting is completely reconstructed during germ cell development of cloned animals, the result of progeny TTFs cloning is also a natural evidence for improving cloning efficiency by H3K27me3 imprinted gene knockout.

Table 4: SCNT embryonic development from enucleated oocytes with various donor cells

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；

H versus h；I versus i；J versus j；K versus k；L versus l:No significant difference；

Δ2represents monoallelic Sfmbt2,Jade1 double deletions；

Δ3represents monoallelic Sfmbt2,Jade1,and Gab1 triple deletions；

B6/PWK represents C57BL/6x PWK/PhJ mice；

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

Table 5: delta 4-embryo under different strategies^3KODevelopment of

Table 6: in vitro development of Nuclear transfer embryos by different culture methods

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

C versus c:p<0.05.

Thinking and discussion

Large fetuses and large placentas were observed in somatic cloning mice (tamashhiro et al, 2002), sheep (Fletcher et al, 2007) and cattle (Smith et al, 2012). It has been reported that overgrowth of the placenta precedes overgrowth of the fetus, so it is likely that a large placenta results in a large fetus. In the present invention, the inventors demonstrated that the h3k27me3 imprinted gene expressed by the biallelic gene significantly hindered the development of SCNT placenta. Due to the natural deletion of H3k27me3 imprinting in the mesoderm-derived somatic cell line, the H3k27me3 imprinting barrier may affect all cloned placentas if somatic cells were used as donors (fig. 4H).

After h3k27me3 imprinted gene single allele in somatic cell donor cells is deleted, the weight of the placenta, the proportion of fetal vessels in a labyrinth layer, the density of the vessels and a cell-free discontinuous area of the placenta are improved well. Moreover, the cloned mice had significant recovery in body weight. Since imprinting LOSs occurs specifically in the placenta, this improvement not only demonstrates that placental h3k27me3 imprinting abnormalities are responsible for large fetuses, but also supports the hypothesis that placental genetic abnormalities in cloned animals may lead to a LOS phenotype.

The h3k27me3 imprinting abnormality of SCNT blastocysts was first discovered by Matoba et al (Matoba et al, 2018). However, the developmental significance of this finding was not determined until the present application. Unlike the classical imprinted gene, its loss in cloning is random, whereas the non-classical imprinted gene imprinting loss is fixed (fig. 1C and 5C). This difference makes H3K27me 3a new type of abnormal imprinting affecting somatic cloned embryos, which explains why the expiration rate increases after deletion of the H3K27me3 imprinted gene.

Since progeny TTFs without H19, IG, or Rasgrf1 deletions were used for cloning, the observed effect was only due to H3K27me3 imprinting deletion. The inventors found that a single-gene single-allele knock-out (Sfmbt 2. delta./+) was very effective in ameliorating placental defects and increasing colony birth rates (albeit to a lesser extent than 4 KO). The h3k27me3 imprinting knockout method may also be widely applied in combination with the gene editing targeted SNP apparent erasing technology.

XIst is reported to exhibit H3K27me3 imprinting patterns, with imprinting silencing in extraembryonic tissues and loss of imprinting in the epiblast. As with the four knocked-out h3k27me3 imprinted genes, the inventors found 2-fold overexpression of Xist in SCNT placenta (fig. 1B). Notably, deletion of Xist single allele in the testicular support cells significantly improved the cloning efficiency from 1.6% to 15.4%. These results indicate that loss of print at Xist is another SCNT epigenetic disorder dependent on h3k27me 3. Recently, several suspected autosomal non-classical imprinting genes were found in human embryos, suggesting that the h3k27me 3-dependent imprinting may also be conserved in mammals that are evolutionarily distant. However, recently it was reported that in human embryos at the 4-8 cell stage, the H3K27me3 modification was extensively erased and Xist failed to survive, a controversial result suggesting that there may be no H3K27me3 imprint in humans. Furthermore, maternal-specific Xist imprinting is not conserved in many species, which may limit the use of Xist knockouts in improving animal cloning efficiency.

Fetal or adult fibroblasts are widely used in important large animal clones, including sheep (Schnieke et al, 1997), cattle (Cibelli et al, 1998), pigs (Onishi et al, 2000) and monkeys (Liu et al, 2018), among others. However, mouse fibroblast cloning efficiency was low compared to other donor cell types. The research result of the inventor shows that the cloning efficiency of both fetal fibroblasts and adult fibroblasts is obviously improved after the h3k27me 3-dependent imprinted gene is deleted. More importantly, the increase in expiration rates for Δ 4 or Sfmbt2 knockouts was significantly higher than for Δ Xist fibroblasts. The inventors not only demonstrated that h3k27me 3-dependent loss of imprinting is an inherent post-implantation epigenetic disorder in scnt-mediated cloning, leading to large placenta and offspring defects, but also demonstrated that modifying h3k27me3 imprinting could be an effective way to improve animal cloning efficiency.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

SEQUENCE LISTING

<110> institute of animal research of Chinese academy of sciences

<120> a somatic cell nuclear transfer method and its application

<130> IDC200253

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<213> Artificial

<220>

<223> ΔXist R

<400> 30

cggccactac tatgagcagg 20

Claims

1. A modified haploid embryonic stem cell wherein the haploid embryonic stem cell is knocked out for a gene selected from the group consisting 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, and any combination thereof;

optionally, additional H19 and IG are knocked out in the haploid embryonic stem cell, and optionally, additional Rasgrf1 is knocked out;

preferably, the haploid embryonic stem cell is knocked out for a gene selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof;

more preferably, in said haploid embryonic stem cell, Sfmbt2 is knocked out, and optionally, a gene selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof;

most preferably, in said haploid embryonic stem cell, a gene or combination of genes selected from the group consisting of:

only Sfmbt2 is knocked out,

alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 were simultaneously knocked out.

2. A method of making the modified haploid embryonic stem cell of claim 1 comprising:

obtaining the modified haploid embryonic stem cell by a gene knockout technique, wherein the gene knockout technique knocks down a gene in the haploid embryonic stem cell selected from the group consisting 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, and any combination thereof;

optionally, the gene knockout technique knocks out additional H19 and IG in the haploid embryonic stem cell, and optionally, knocks out additional Rasgrf 1;

preferably, the gene knockout technique knocks out a gene in a haploid embryonic stem cell selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof;

more preferably, the gene knockout technique knocks out Sfmbt2 in a haploid embryonic stem cell, and optionally, knockouts selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof;

most preferably, the gene knockout technique knocks out a gene or combination of genes in the haploid embryonic stem cell selected from the group consisting of:

only Sfmbt2 is knocked out,

or, simultaneously knocking out Sfmbt2, Jade1, Gab1 and Smoc 1;

or preferably, the gene knockout technique is a gene knockout technique using CRISPR.

3. A somatic cell, wherein the somatic cell has been single-allele knocked-out of a gene selected from the group consisting 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, and any combination thereof;

optionally, additional H19 and IG in the somatic cell are single-allele knocked-out, and optionally, additional Rasgrf1 is single-allele knocked-out;

preferably, the somatic cell is single-allele knocked-out for a gene selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof;

more preferably, in said somatic cell, Sfmbt2 is single-allele knocked out, and optionally, a gene selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof;

most preferably, in said somatic cell, a gene or combination of genes selected from the group consisting of:

only Sfmbt2 is knocked out,

alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 were simultaneously knocked out;

or preferably, the somatic cell is derived from a mammal (preferably a non-human mammal);

more preferably, the mammal is a mouse, sheep, cow, pig or monkey;

or preferably, the somatic cell is a fibroblast;

more preferably, the fibroblast is a fetal or adult fibroblast (e.g., a tip fibroblast).

4. A method of making the somatic cell of claim 3, comprising:

(1) providing the modified haploid embryonic stem cell of claim 1,

(3) culturing and developing the first reconstituted embryo into a fetus;

(4) isolating and obtaining the somatic cells from the fetus;

preferably, in step (3), the first reconstituted embryo is cultured to develop into a fetus by using a tetraploid blastocyst;

more preferably, the method comprises:

1) providing the modified haploid embryonic stem cell of claim 1,

6) isolating and obtaining the somatic cells from the fetus;

preferably, the surrogate mother uterus is a mammalian (preferably non-human mammal) surrogate mother uterus;

more preferably, the surrogate mother uterus is a mouse, sheep, cow, pig or monkey mother uterus;

alternatively, preferably, the tetraploid blastocyst is obtained by an electrofusion or chemical fusion method.

5. Use of the somatic cells of claim 3 or obtained by the method of claim 4 for nuclear transfer.

6. A method of nuclear transfer of somatic cells, which uses donor somatic cells for nuclear transfer, wherein the donor somatic cells are as defined in claim 3;

preferably, the method comprises:

b-1) activating the third reconstructed embryo;

alternatively, the method comprises:

b-2) activating the fourth reconstructed embryo.

7. Use of the modified haploid embryonic stem cell of claim 1 or the somatic cell of claim 3 in the manufacture of a medicament for treating a disease associated with somatic cell nuclear transfer;

preferably, the disease associated with somatic cell nuclear transfer is fetal macrosomia syndrome;

preferably, the disease associated with somatic cell nuclear transfer is a large placenta and/or a large fetus;

preferably, the disease associated with somatic cell nuclear transfer is overweight newborn fetus in gestational diabetic mammals;

preferably, the fetal presenting syndrome is mammalian (preferably non-human mammalian) fetal presenting syndrome;

preferably, the mammal is a mouse, sheep, cow, pig or monkey.

8. A method of treating a disease associated with somatic cell nuclear transfer, comprising: performing nuclear transfer using donor somatic cells, wherein the donor somatic cells are as defined in claim 3;

preferably, the mammal is a mouse, sheep, cow, pig or monkey;

preferably, the method comprises:

b-1) activating the third reconstructed embryo;

alternatively, the method comprises:

b-2) activating the fourth reconstructed embryo.

9. An animal, wherein said animal has been single allele knocked out for a gene selected from the group consisting 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, and any combination thereof;

optionally, additional H19 and IG are single-allele knockouts in the animal, and optionally, additional Rasgrf1 is single-allele knockouts;

preferably, the animal is single-allele knocked-out for a gene selected from the group consisting of: sfmbt2, Jade1, Gab1, Smoc1, and any combination thereof;

more preferably, in said animal Sfmbt2 is single allele knockout, and optionally, a gene selected from the group consisting of: jade1, Gab1, Smoc1, and any combination thereof;

most preferably, in said animal, a gene or combination of genes selected from the group consisting of:

only Sfmbt2 is knocked out,

alternatively, Sfmbt2, Jade1, Gab1 and Smoc1 were simultaneously knocked out;

or preferably, the animal is a mammal (preferably a non-human mammal);

more preferably, the mammal is a mouse, sheep, cow, pig or monkey.

10. A method of making the animal of claim 9, comprising:

culturing the activated third or fourth reconstructed embryo obtained by the method of claim 7;