Gene for promoting SCNT embryonic development, development fate system and construction method

Abstract

The invention belongs to the technical field of somatic cell nuclear transfer, and discloses a gene for promoting SCNT embryonic development, a development fate system and a construction method. The gene for promoting the SCNT embryonic development is an RNA (Ribonucleic Acid) interference fragment after Setdb2 knockdown or a gene after Shpry overexpression. The RNA interference fragment with the Setdb2 knocked down comprises a knock-down site Setdb2-1597, and the knock-down sequence of the RNA interference fragment is SEQIDNO: 13 and SEQIDNO: 14. According to the invention, a system for tracking SCNT embryo blastocyte development fate is established through pinhole culture, SCNT embryo transcriptome databases with different developmental capacities are constructed through single cell transcriptome sequencing, and regulation and control effects of Setdb2 and Shpry on SCNT embryo development are clarified through knock-down or overexpression. The method has important scientific guiding significance on improvement and application of SCNT efficiency.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of somatic cell nuclear transfer, and particularly relates to a gene for promoting development of SCNT embryos, a development fate system and a construction method. BACKGROUND

[0002] Somatic cell nuclear transfer (SCNT) is a reproductive biotechnology that can reprogram differentiated somatic cells into totipotent embryos, and has a wide application prospect in endangered species protection and therapeutic cloning. However, the low efficiency and high abnormal phenotype rate of SCNT seriously hinder the wide application of the technology. Solving the problem of incomplete or incorrect epigenetic modification of SCNT will help to improve the cloning efficiency.

[0003] Recent studies have found that SCNT embryos often exhibit abnormal epigenetic modification, especially abnormal histone methylation, which reduces the developmental potential of SCNT embryos. For example, histone H3 lysine 9 trimethylation (H3K9me3) of the donor cell genome is considered to be a major obstacle to SCNT reprogramming, and removal of H3K9me3 can significantly improve the efficiency of SCNT.

[0004] Although a lot of important progress has been made in the study of SCNT embryo development, these studies are based on whole-level research and do not analyze and study individual blastomeres, which cannot reflect the actual situation. Single-cell transcriptome sequencing provides a new way to explore the real reprogramming factors related to SCNT embryo development. With the development and improvement of low sample sequencing technology, people can detect more detailed whole-genome epigenetic modification maps in SCNT embryos, further revealing the defects in the epigenetic reprogramming of SCNT embryos and providing clues to improve cloning efficiency.

[0005] The well-of-well system (WOW) realizes multidimensional cell culture and analysis by culturing cells in microwells, and provides an important method for tissue engineering, drug screening, disease research and cell function research.

[0006] At present, it has been found that the development of pig SCNT embryos is mostly blocked before the 4-cell stage, but there is no research on the mining and elucidation of regulatory factors for pig SCNT embryos with different developmental potential.

[0007] Through the above analysis, the problems and defects of the prior art are: somatic cell nuclear transfer is a reproductive biotechnology that can reprogram differentiated somatic cells into totipotent embryos, and has a wide application prospect in endangered species protection and therapeutic cloning. However, the low efficiency and high incidence of abnormal phenotype of SCNT seriously hinder the wide application of the technology. At present, it is found that the development of pig SCNT embryos is mostly blocked before the 4-cell stage, but there is no research on the mining and elucidation of regulatory factors for pig SCNT embryos with different developmental potential. SUMMARY

[0008] In order to overcome the problems in the related art, the present application discloses a gene for promoting SCNT embryo development, a development fate system and a construction method, which specifically relates to the establishment and regulation of a pig cloning embryo development capacity tracking system. The purpose of the present application is to fully mine and screen the real regulatory factors related to the development of SCNT embryos by establishing a small hole culture system and tracking the development fate of each SCNT embryo, combined with single cell transcriptome sequencing, and then improving the SCNT efficiency by regulating these factors.

[0009] The technical solution is as follows: a gene for promoting SCNT embryo development, which is an RNA interference fragment after Setdb2 knockdown, or a gene after overexpression. Shprh The gene after overexpression is SEQ ID NO: 1.

[0010] The RNA interference fragment after Setdb2 knockdown includes: a knockdown site Setdb2-497, and a knockdown sequence of SEQ ID NO: 9 and SEQ ID NO: 10.

[0011] The RNA interference fragment after Setdb2 knockdown includes: a knockdown site Setdb2-1170, and a knockdown sequence of SEQ ID NO: 11 and SEQ ID NO: 12.

[0012] The RNA interference fragment after Setdb2 knockdown includes: a knockdown site Setdb2-1597, and a knockdown sequence of SEQ ID NO: 13 and SEQ ID NO: 14.

[0013] Shprh The gene after overexpression Shprh The gene after overexpression is SEQ ID NO: 1, which is used for down-regulation of γ-H2AX.

[0014] Another object of the present application is to provide a system for tracking the development fate of blastomeres of SCNT embryos, which is built by optimizing the small pore culture system of cloned embryos, blastomere separation technology and development tracking, and based on the system for tracking the development fate of blastomeres of SCNT embryos, genes related to the development of SCNT embryos are mined and screened.

[0015] The genes related to the development of SCNT embryos are RNA interference fragments after Setdb2 knockdown, or genes after overexpression. Shprh

[0016] Another object of the present application is to provide a construction method of a system for tracking the development fate of blastomeres of SCNT embryos, which is used for the construction of the system for tracking the development fate of blastomeres of SCNT embryos, and the construction method comprises the following steps: The construction of the system for tracking the development fate of blastomeres of SCNT embryos specifically comprises the following steps: S1, collecting and maturing culturing oocytes, primary cell culturing, cell passage, cryopreservation and recovery, somatic cell nuclear transfer, sperm preparation, and in vitro fertilization; S2, small pore culture system and blastomere separation of cloned embryos are further performed; S3, statistical analysis, single factor variance analysis is performed on the development of cloned embryos by using SPSS statistical software, relative integral optical density is analyzed by using ImageJ, and gene expression is analyzed by using 2 −ΔΔCT Method; the experimental results are expressed as mean ± standard error.

[0017] In step S2, the small pore culture system comprises the following steps: selecting cloned embryos with uniform blastomere division, placing the cloned embryos in a protease K solution, cutting the zona pellucida of the SCNT embryos with a needle, separating the blastomeres, placing one of the blastomeres in a PE tube after washing with PBS for cryopreservation, and placing the other homologous blastomere in a small pore culture plate and marking.

[0018] In step S2, the distance between the small pores is 500 µm. In step S2, the blastomere separation time of the cloned embryos is 30 h.

[0019] In combination with all the technical solutions described above, the present application has the following beneficial effects: ​The application aims to establish a small pore culture system and a method for tracking the development fate of each SCNT embryo, combine single cell transcriptome sequencing, mine and screen the real development-related regulatory factors of the SCNT embryo, and then realize the totipotency stage transition of the SCNT embryo and improve the SCNT efficiency through regulating these factors.

[0020] The main positive effect results of the application are as follows: the small pore culture system has no influence on the cleavage rate and blastocyst rate of embryos, and the 500 mu m small pore spacing is the best; the early embryo cleavage and cleavage ball development fate results show that the cleavage rate of the SCNT embryo gradually and significantly increases at 24 h, 30 h and 36 h, and the 2-cell rate is the highest at 30 h; compared with the parthenogenetic embryo, the development of the SCNT embryo is slowed down, and the 4-cell rate is significantly reduced at 30 h and 36 h; the tracking results of the 2-cell to 4-cell transition of the SCNT embryo show that the subsequent development synchronization rate of the 2-cell cleavage ball after separation is above 86.6%, and the cleavage ball development fate tracking system of the SCNT embryo is established.

[0021] The single cell transcriptome results of the SCNT embryo cleavage ball with different development fates show that, compared with the development arrest cleavage ball, there are 31 obviously up-regulated genes and 110 significantly down-regulated genes in the normal cleavage cleavage ball, GO function and pathway analysis shows that, Setdb2 and Shprh are involved in histone modification and DNA damage.

[0022] In the development process of the SCNT embryo, the histone modification results show that, compared with the in vitro fertilization embryo, the H3K9me3 of the SCNT embryo is significantly up-regulated at 1-cell, 2-cell and 4-cell; compared with the development arrest SCNT embryo, the H3K9me3 of the normally developed SCNT embryo is significantly down-regulated.

[0023] Setdb2 The H3K9me3 of the development arrest SCNT embryo cleavage ball is significantly up-regulated, and the H3K9me3 of the SCNT embryo is Setdb2 The knockdown results show that siRNA-1597 significantly reduces the expression of Setdb2 , and promotes the 4-cell development of the SCNT embryo; Setdb2 After knockdown, the H3K9me3 is significantly reduced, which indicates that Setdb2 is not conducive to the development of the SCNT embryo, but after knockdown, the totipotency transition of the SCNT embryo is promoted by improving the H3K9me3 modification.

[0024] Shprh The H3K9me3 of the development arrest SCNT embryo is significantly down-regulated, and the H3K9me3 of the SCNT embryo is Shprh The overexpression results show that ShprhThe development efficiency of the SCNT embryo is obviously improved after overexpression, and gamma-H2AX is significantly down-regulated, which indicates that Shprh The development of the SCNT embryo is promoted by reducing DNA damage.

[0025] The present application establishes a system for tracking the development fate of blastomeres of the SCNT embryo by small pore culture, constructs a transcriptional database of the SCNT embryo with different development capacities by single-cell transcriptome sequencing, and determines the regulation effect of Setdb2 and Shprh on the development of the SCNT embryo by knockdown or overexpression. Setdb2 and Shprh The present application has important scientific guiding significance for improving the efficiency of the SCNT and application.

[0026] The present application determines that Setdb2 and Shprh have the regulation effect on the development of the cloned embryo by screening differential genes through single-cell transcriptome sequencing on the development-arrested or normally developed cloned embryo.

[0027] The advantages of the present application further include: a system for tracking the development fate of blastomeres of the SCNT embryo is established by optimizing the small pore culture system of the cloned embryo, blastomere separation technology and development tracking; the key genes related to the development potential of the cloned embryo are screened by leading single-cell transcriptome sequencing; Setdb2 is knocked down to realize the significant decrease of H3K9me3 and promote the development of the SCNT embryo to 4-cell; Shprh is overexpressed to realize the significant down-regulation of gamma-H2AX and the significant increase of the development of the SCNT embryo to 4-cell, and no related literature is reported. BRIEF DESCRIPTION OF DRAWINGS

[0028] The drawings incorporated into the specification and constituting a part of the specification show embodiments consistent with the present disclosure and, together with the specification, serve to explain the principles of the present disclosure; Figure 1 is a flowchart for constructing a system for tracking the development fate of blastomeres of the SCNT embryo provided by the embodiments of the present application; Figure 2 is a development situation diagram of the embryo in different spacing small pores provided by the present application; Figure 3 is a blastomere development situation diagram provided by the present application; Figure 4 is a blastomere development situation diagram provided by the present application; Figure 5 is a differential gene statistical diagram provided by the present application; Figure 6 is a gene molecular function analysis diagram provided by the present application; Figure 7 is a differential gene expression pattern diagram of the cloned embryo with different development fates provided by the present application; Figure 8is the H3K9me3 immunofluorescence chart of the in vitro fertilization embryo and the SCNT embryo at different development stages provided by the present application; Figure 9 is the H3K9me3 immunofluorescence chart of the development arrest embryo and the normal development embryo provided by the present application; Figure 10 is the gamma-H2AX immunofluorescence chart of the development arrest embryo and the normal development embryo provided by the present application; Figure 11 is the mRNA expression chart after Setdb2 knockdown provided by the present application; Figure 12 is the SCNT embryo development chart after Setdb2 knockdown provided by the present application; Figure 13 is the H3K9me3 immunofluorescence chart of the 4-cell SCNT embryo after Setdb2 knockdown provided by the present application; Figure 14 is the SCNT embryo development chart after overexpression of Shprh provided by the present application; Figure 15 is the gamma-H2AX immunofluorescence chart of the 4-cell SCNT embryo after overexpression of Shprh provided by the present application. DETAILED DESCRIPTION

[0029] In order to make the above objectives, characteristics and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application are described in detail below in combination with the drawings. In the following description, a large number of specific details are set forth in order to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0030] The innovation point of the present application is that the present application proposes a specific process, a specific culture method, a specific gene and a specific regulation. The present application builds a tracking system for the development fate of the cleavage ball of the SCNT embryo through the small pore culture, the cleavage ball separation technology and the development tracking system. The key gene related to the development potential of the cloned embryo is screened by leading single cell transcriptome sequencing; the SCNT embryo 4-cell development is promoted by knocking down Setdb2 for the first time. The overexpression of Shprh to promote the development of the cloned embryo is also not reported in the related literature.

[0031] Example 1, a gene for promoting the development of the SCNT embryo, which is the RNA interference fragment after Setdb2 knockdown, or Shprh the gene after overexpression.

[0032] Exemplarily, the gene for promoting the development of the SCNT embryo is Setdb2. ShprhThe gene is cloned by cDNA, a construct in vitro transcription vector is constructed, mRNA is transcribed in vitro, microinjection is carried out, and overexpression is realized Shprh .

[0033] The RNA interference fragment after Setdb2 knockdown includes: a knockdown site Setdb2-497, and the knockdown sequence is SEQ ID NO: 9 and SEQ ID NO: 10.

[0034] The RNA interference fragment after Setdb2 knockdown includes: a knockdown site Setdb2-1170, and the knockdown sequence is SEQ ID NO: 11 and SEQ ID NO: 12.

[0035] The RNA interference fragment after Setdb2 knockdown includes: a knockdown site Setdb2-1597, and the knockdown sequence is SEQ ID NO: 13 and SEQ ID NO: 14.

[0036] Shprh The gene after overexpression is SEQ ID NO: 1; and the gene is used for down-regulation of gamma-H2AX.

[0037] It can be understood that Shprh Overexpression refers to that the cDNA sequence of the gene is amplified, and then subjected to steps such as enzyme digestion and ligation, so as to finally construct an in vitro transcription plasmid, which is transcribed into mRNA by an in vitro transcription system, and still is the Shprh Gene sequence after microinjection, so as to realize gene overexpression; the overexpression mode is realized based on an in vitro transcription system and microinjection, and is different from a liposome transfection method after construction of a plasmid.

[0038] The application provides a tracking SCNT embryo blastomere development fate system for screening the gene for promoting SCNT embryo development, which is built by optimizing a cloned embryo small pore culture system, a blastomere separation technology and development tracking, and based on the tracking system, single cell transcriptome sequencing is combined to mine and screen the gene for promoting SCNT embryo development related to SCNT embryo development.

[0039] The gene for promoting SCNT embryo development related to SCNT embryo development is a RNA interference fragment after Setdb2 knockdown, or is Shprh A gene after overexpression.

[0040] Another purpose of the application is to provide a construction method of a tracking SCNT embryo blastomere development fate system, which is used for construction of the tracking SCNT embryo blastomere development fate system, and the construction method comprises the following steps: constructing the tracking SCNT embryo blastomere development fate system by optimizing a cloned embryo small pore culture system, a blastomere separation technology and development tracking; AsFigure 1 As shown, the system for tracking the development fate of SCNT embryo blastomere specifically comprises: S1, collecting and maturing culture of oocytes, primary cell culture, cell passage, cryopreservation and recovery, somatic cell nuclear transfer, sperm preparation, in vitro fertilization; S2, further performing a small pore culture system, and separating blastomeres of the cloned embryo; S3, statistical analysis, using SPSS statistical software for single factor variance analysis on the development of the cloned embryo, using ImageJ analysis for relative integrated optical density, and using 2 −ΔΔCT method for gene expression analysis; the experimental results are expressed as mean ± standard error.

[0041] Step S2, further performing a small pore culture system, which comprises: selecting cloned embryos with uniform blastomere division, placing the cloned embryos in a protease K solution, separating the blastomeres with a needle, placing one of the blastomeres in a PBS solution and then moving it into a PE tube for cryopreservation, and placing the other blastomere into a small pore culture plate and marking it.

[0042] In step S2, the small pore culture system, the distance between the small pores is 500 µm. In step S2, the blastomere separation of the cloned embryo, the blastomere separation time of the SCNT embryo is 30 h.

[0043] Through the above experiment, it can be known that the system for tracking the development fate of SCNT embryo blastomere is built by optimizing the small pore culture system of the cloned embryo, the blastomere separation technology and the development tracking.

[0044] Compared with the development-arrested SCNT embryo, the H3K9me3 of the normally developed embryo is significantly down-regulated.

[0045] Setdb2 is significantly up-regulated in the development-arrested SCNT embryo blastomere, and after knockdown, the H3K9me3 is significantly decreased, and the 4-cell development of the SCNT embryo is promoted.

[0046] Shprh is significantly down-regulated in the development-arrested SCNT embryo, and after overexpression of Shprh, the γ-H2AX is significantly down-regulated, and the 4-cell development of the SCNT embryo is significantly improved.

[0047] As can be known from the above examples, SCNT has broad application prospects in the fields of livestock, biological medicine and human medicine, but the low cloning efficiency limits its application. The cloned embryo development can be improved by regulating genes in the present application, which can accelerate the propagation of excellent livestock, the cultivation of new varieties, the production of precious medicines and the preparation of humanized organs, and has huge commercial value.

[0048] In the field of animal cloned embryo development, there is no report on improving the efficiency of cloned embryo development by regulating the two genes of the application. The two genes are identified in the application, and the efficiency of somatic cell nuclear transfer is improved by regulating the epigenetic modification of cloned embryos, which is the first report and has important significance for promoting the application of somatic cell nuclear transfer technology.

[0049] The low efficiency of somatic cell nuclear transfer limits its application. Scientists have improved embryo culture medium, epigenetic modification and gene expression to a certain extent to improve embryo development, but the efficiency is still not high. The application is based on development arrest and normal development of cloned embryos, and two differential genes are identified by single cell transcriptome sequencing, and the development of cloned embryos is improved by regulation, which solves the problem of low efficiency of somatic cell nuclear transfer to a certain extent.

[0050] The application comprehensively uses somatic cell nuclear transfer, embryo splitting, small pore culture, single cell transcriptome sequencing, gene expression regulation, etc., overcomes technical bias, and realizes the improvement of cloned embryo development ability.

[0051] In the above embodiments, the description of each embodiment has its own emphasis, and the parts not described or recorded in a certain embodiment can be referred to the related description of other embodiments.

[0052] To further illustrate the related effects of the embodiments of the application, the following experiments are carried out.

[0053] Most of the development of pig SCNT embryos will be arrested before the 4-cell stage, and the existing technology has not yet explored and clarified the role of regulatory factors for pig SCNT embryos with different development potential. The application intends to establish a small pore culture system and a method of tracking the development fate of each SCNT embryo, combined with single cell transcriptome sequencing, to fully explore and screen the real regulatory factors related to the development of SCNT embryos, and then improve the efficiency of SCNT by regulating these factors.

[0054] Test Example 1, test one to establish a system for tracking the development fate of cloned embryo blastomeres.

[0055] 1. The current SCNT embryo culture system is not perfect, and there is no effective system to track the development of each cloned embryo. Therefore, the application intends to establish a small pore culture system to track the development of each embryo, and at the same time, through the separation and detection of blastomere development synchrony, a system for tracking the development fate of cloned embryo blastomeres is established.

[0056] 2. Test materials.

[0057] 2.1 Test materials The pig ovaries are derived from Qingdao Wanfu Group.

[0058] 2.2 Main reagents: HEPES buffer, L-cysteine, penicillin, streptomycin, epidermal growth factor, luteinizing hormone, sodium pyruvate, follicle stimulating hormone and nuclear dye Hoechst 33342 were purchased from Sigma Company, and the basic medium TCM-199 was provided by Gibico Company.

[0059] 3. Test method.

[0060] 3.1 Oocyte collection and maturation culture. Oocytes were obtained from the ovaries of pigs, and usually follicles with a diameter of 3-8 mm were selected. The follicular fluid was gently aspirated, and the cumulus-oocyte complex containing more than three layers of granulosa cells was selected from the follicular fluid. The selected cumulus-oocyte complex was cultured in the maturation medium. After 42 h of culture, the oocytes were screened by hyaluronidase solution for 3 min.

[0061] 3.2 Primary cell culture. Pig ear samples were taken, and multiple tissue blocks of about 0.5 cm x 0.5 cm were cut and added to 3 mL trypsin solution for digestion. 3 mL culture medium containing 10% serum and 1% double antibody was added to each culture dish to terminate the trypsin digestion reaction. After centrifugation to remove the supernatant, the cells were resuspended in culture medium for culture.

[0062] 3.3 Cell passage, freezing and recovery. Cell passage: the original culture medium was discarded, 1 mL trypsin was added to the dish, followed by termination of digestion, resuspension of the cells at the bottom of the tube, and continued culture at 37°C in a 5% CO2 incubator.

[0063] Cell freezing: remove the DMEM culture medium in the dish, add 1 mL trypsin, and terminate the digestion. Resuspend the cells with freezing medium containing 90% fetal bovine serum and 10% DMSO, and transfer to liquid nitrogen for storage.

[0064] Cell recovery: the frozen tube stored in liquid nitrogen was taken out and quickly thawed in a 37°C water bath, the cells were resuspended in DMEM culture medium, and transferred to a culture dish for continued culture.

[0065] 3.4 Somatic cell nuclear transfer. Prepare 200 μL of a shuttle operation drop and cover it with paraffin oil. Place the operation drop on the micro-operation table, and add oocytes and somatic cells for nuclear transfer operation. After the oocytes are enucleated, the enucleated oocytes are injected to form reconstructed embryos, which are activated and cultured. The cleavage rate is observed after 48 h, and the blastocyst rate is observed after 156 h.

[0066] 3.5 Sperm preparation. Add about 3 times sperm diluent of semen, mix well, and place at room temperature for 6 h, then store in a 17 ℃ constant temperature refrigerator, and mix every 12 h.

[0067] 3.6 In vitro fertilization. Select mature oocytes that have discharged the first polar body, and transfer to pre-equilibrated fertilization liquid droplets at a density of 30-35 per droplet; adjust the sperm density to 2-5 x 10 5 / mL with mTbm liquid, and add to the culture for 6 h, then culture in embryo culture liquid, count the cleavage rate at 48 h after fertilization, and evaluate the blastocyst rate at 156 h.

[0068] 3.7 Small pore culture system. Steel needles (needle tip diameter 300 µm) are used to vertically pierce uniform micro-holes in a four-well plate, 48 micro-holes are pierced in each well, and 400 µL of embryo culture liquid is added to the treated four-well plate.

[0069] 3.8 Cloned embryo blastomere separation. Select cloned embryos with uniform blastomere division, place in a protease K solution, separate the blastomeres with a needle, and after washing in PBS, one of the blastomeres is moved to a PE tube for storage, and the other homologous blastomere is moved to a small pore culture plate and labeled, and the subsequent development is observed.

[0070] 3.9 Statistical analysis. All statistical analysis is at least three independent repeats, the development of cloned embryos is statistically analyzed using SPSS statistical software one-way ANOVA, the relative integrated optical density is analyzed using ImageJ, and the gene expression is analyzed using 2 −ΔΔCT Method. The test results are "mean ± standard error (MEAN ± SEM)", and the difference of related data is considered significant when P <0.05.

[0071] 4. Test results.

[0072] 4.1 Effect of different small pore distances on cloned embryo development. As Figure 2 embryos in different small pore distances develop, Figure 2 A figure in which embryos in different small pore distances develop; B figure is the cleavage rate of embryos cultured in different distance small pores; C figure is the blastocyst rate of embryos cultured in different distance small pores.

[0073] It can be seen that different small pore distances do not affect the cleavage rate of embryos; the blastocyst rate results show that, compared with the control group, the 500 µm small pore distance is better, therefore, the 500 µm small pore diameter is suitable for tracking embryo development.

[0074] 4.2 Determination of the optimal cleavage time point of cloned embryos. As Figure 3Embryo cleavage at different time points, Figure 3 Figure A is the cleavage state of parthenogenetic embryos and SCNT embryos at different time points; Figure B is the cleavage rate of parthenogenetic embryos and SCNT embryos at different time points; Figure C is the proportion of 2-cell of parthenogenetic embryos and SCNT embryos; Figure D is the proportion of each stage of parthenogenetic embryos and SCNT embryos.

[0075] It was found that the cleavage rate of SCNT embryos gradually increased significantly, and the 2-cell rate was the highest at 30 h; compared with parthenogenetic embryos, SCNT embryos developed more slowly (Figure C, P <0.05). The experiment will separate the blastomeres at 30 h and track the development to 4-cell. In summary, the cleavage rate of SCNT embryos is the highest at 30 h, which helps the separation of blastomeres and subsequent tracking.

[0076] 4.3 Blastomere development synchronization rate, such as Figure 4 Blastomere development, Figure 4 Figure A is the development state of SCNT embryo blastomeres; Figure B is the development synchronization rate 24 h after the separation of blastomeres; Figure C is the separation state of blastomeres. In Figure C, 1-1 represents that both homologous blastomeres are developmentally arrested; 1-2 represents that one of the homologous blastomeres is developmentally arrested; 2-2 represents that both homologous blastomeres develop; 4-4 represents that both homologous blastomeres divide twice.

[0077] The 2-cell stage SCNT embryo blastomeres were separated at 30 h, and the development of the blastomeres in two different plate wells was observed. It was found that the synchronization rate was more than 86.6%, and the homologous blastomeres that could continue to develop accounted for more than 63%. This indicates that the synchronization rate of SCNT embryo blastomere development fate is high.

[0078] 5. In vitro embryo development is affected by many factors, such as embryo density during culture (Lane and Gardner 1992, Kato and Tsunoda 1994). In the small pore culture system, micropores are designed at the bottom of the culture dish, which solves the problem of embryo displacement that may occur in traditional microdrop culture. The WOW system has significant advantages in improving blastocyst rate and embryo quality, and more importantly, the small pore culture system can track the development of each SCNT embryo. In the present invention, four small pore culture systems with different small pore spacings were made, and the embryos were placed in the small pores for culture after SCNT. The research results show that the small pore culture system does not significantly affect embryo development, but the blastocyst rate is the highest when the small pore spacing is 500 µm. Therefore, the small pore culture system with a small pore spacing of 500 µm is selected for subsequent experiments.

[0079] The first cleavage time of SCNT embryos is usually delayed 2-4 h compared with naturally fertilized embryos, which may be related to the lag of nuclear reprogramming. In the present invention, the present invention compared the proportion of each state at different time points of parthenogenetic embryos and SCNT embryos, and found that the cleavage time of SCNT embryos was later than that of parthenogenetic embryos, and the 2-cell rate of SCNT embryos was the highest at 30 h, which may be due to the longer reprogramming time required by SCNT embryos. Therefore, the present invention selected the separation of blastomeres at 30 h.

[0080] Homologous blastomeres have consistency in genetic material. In this experiment, the present invention separated SCNT embryos and observed the development of blastomeres. The present invention found that the subsequent development synchronization rate of blastomere separation was above 86.6%, thus the present invention could prove that the developmental fate of homologous blastomeres had correlation. There is also experimental evidence that shows that after splitting the 2-4 cell stage embryo of mammals, each blastomere can develop into a normal individual, supporting the consistency of early embryonic blastomere development synchronization (Sheikholslamietal. 2008).

[0081] 6. In this experiment, the present invention aims to explore the effect of small pore culture system on SCNT embryo development, and to find the best cleavage time of SCNT embryos and the synchronization rate of homologous blastomeres. The results show that the small pore culture system does not significantly affect embryo development, but the blastocyst rate is the highest when the small pore distance is 500 µm; the 2-cell rate of SCNT embryos is the highest at 30 h, which is suitable for blastomere separation; the development synchronization rate of 2-cell cloned embryo blastomeres cultured in small pores is high. In summary, the present invention has built a system to track the cleavage rate and developmental fate of individual cloned embryos.

[0082] Test Example 2, Test Two, Mining of Key Regulatory Factors for Cloned Embryo Development.

[0083] 1. Cloning technology, as a cutting-edge bioengineering means, shows broad application prospects in the fields of animal husbandry, drug research and development, and endangered animal protection, etc. However, the development arrest problem of SCNT embryos has become a key bottleneck restricting the development of this technology. With the wide application of single-cell sequencing technology, it provides support for finding the regulatory factors of SCNT embryo development. In this test, single-cell transcriptome sequencing technology was used to sequence and analyze the development arrested SCNT embryos and the SCNT embryos developed to 4-cell, and the factors regulating the development of SCNT embryos were screened, and their ability to regulate embryo development was verified.

[0084] 2. Test materials. Same as part of Test One.

[0085] 3. Test method.

[0086] 3.1 Real-time PCR.

[0087] 3.1.1 RNA isolation and purification. Total RNA was extracted from different blastomeres of SCNT embryos with RNeasy Mini Kit according to the manufacturer's standard protocol, and high purity RNA samples were obtained.

[0088] 3.1.2 cDNA synthesis. 1 μL total RNA sample was taken to construct a 20 μL reaction system with 5x EvoM-MLV RT Master Mix reverse transcription premix. The obtained cDNA product was stored at -20 ℃ for standby.

[0089] 3.1.3 Quantitative analysis of gene expression. Based on SYBR Green fluorescence quantitative technology, amplification was performed using ABI real-time fluorescent quantitative PCR detection system. The primers were designed by Primer Premier 5.0 software and synthesized by Shanghai Shengong Biological Engineering Co., Ltd. All samples were repeated three times, and the relative expression of genes was calculated by 2 −ΔΔCT method.

[0090] Table 1 Primer sequences for qRT-PCR

[0091] 3.2 Single-cell transcriptome sequencing. Single-cell sequencing was entrusted to Annuo, based on Illumina sequencing platform, by extracting mRNA from development-arrested and successfully developed SCNT embryo blastomeres, analyzing the differentially expressed mRNA of development-arrested and successfully developed embryos, performing functional annotation, and performing GO function and KEGG analysis of differential genes.

[0092] 4. Results.

[0093] 4.1 The statistical chart of differentially expressed genes is shown in Figure 5 ; blue represents the number of up-regulated DEGs, yellow represents the number of down-regulated DEGs, and cyan represents the total number of genes.

[0094] Gene differential expression analysis was performed using DESeq2 to compare the treatment group and the reference group, and genes with |log2Ratio|≥1 and p<0.05 were selected as significantly differentially expressed genes. Among them, there were 31 significantly up-regulated genes and 110 significantly down-regulated genes in the normally developed embryos compared with the development-arrested embryos.

[0095] 4.2 Molecular function analysis of differential genes, as shown in Figure 6 gene molecular function analysis chart; From the perspective of molecular function, the differentially expressed genes of SCNT embryo developmental arrest are enriched in RNA polymerase II (Pol II) cis-regulatory region binding, zinc ion binding, ubiquitin-protein ligase activity, and DNA-binding transcription repressor activity.

[0096] 4.3 Verification of differentially expressed genes, such as Figure 7 Differential gene expression patterns in cloned embryos with different developmental fates; wherein developmental block: SCNT embryos that develop to the 2-cell stage; 4-cell: SCNT embryos that develop normally.

[0097] Two genes with significant differential expression were screened. Among them, SEQ ID NO: 1, Shprh Gene series, SEQ ID NO: 2, Setdb2 Gene series, SEQ ID NO: 2, as follows: For example, set the 2-cell developmental arrest SCNT embryo as the control group, and the SCNT embryo that can develop to the 4-cell stage as the experimental group, and perform qRT-PCR detection on the two groups of oocytes, and analyze the results, Shprh There is a significant upward trend, Setdb2 There is a significant downward trend.

[0098] 5. Abnormal negative regulation of cell differentiation can also cause SCNT embryo growth to stop. Key negative regulatory factors, such as the PRC2 complex (Chen et al. 2020b), regulate H3K27me3 modification, which inhibits the expression of differentiation genes. The main functions of cis-regulatory elements include regulating transcription initiation, enhancing or inhibiting expression, spatiotemporal-specific regulation, and chromatin structure regulation. The transcriptional activity of RNAPolII depends on the binding of transcription factors to cis-regulatory elements, recruiting co-activators or chromatin remodeling complexes, and regulating gene-specific expression (Zou et al. 2022a). The epigenetic memory of donor cell nuclei, such as abnormal histone modification, hinders the binding of PolII and transcription factors, which in turn leads to embryo failure.

[0099] E3 ubiquitin ligases, such as Shprh, mediate its ubiquitination degradation or regulation by recognizing specific substrates. In SCNT embryos, E3 ligases such as UBR5 fail to degrade donor cell-specific repressor proteins (Tsai et al. 2023) (such as HDACs), leading to persistent silencing of pluripotency genes, thereby inhibiting the development of SCNT embryos. In SCNT embryos, PRC2 complexes, such as EZH2 (Liu and Yang 2023), can be abnormally active in donor cell nuclei, maintaining H3K27me3 modification in the promoter region of pluripotency genes, hindering the activation of pluripotency genes in SCNT embryos.

[0100] In summary, by analyzing the differential genes, an analysis system at the gene level for SCNT embryo development was established, and the key genes for embryo reprogramming disorders were accurately obtained, providing theoretical guidance for optimizing the SCNT method, screening cell reprogramming factors, and developing new epigenetic regulation tools, and ultimately improving the development efficiency of SCNT embryos.

[0101] 6. Gene GO function and pathway significance analysis found that differential genes were widely involved in processes such as transcriptional regulation of RNA polymerase II, protein ubiquitination, and regulation of gene expression, among which Shprh and Setdb2 potentially regulate the differential development of cloned embryos.

[0102] Test Example 3, Test Three, Effect of Histone Modification on Cloned Embryo Development.

[0103] 1. In order to establish a histone modification evaluation system for SCNT embryos, the role of histone in SCNT embryos was determined. In this experiment, the present application systematically analyzed the dynamic pattern of histone modification (H3K9me3) and γ-H2AX expression in pig SCNT embryos during pre-implantation embryo development, compared with in vitro fertilized embryos, to find the differences in histone modification between SCNT embryos and fertilized embryos. At the same time, the present application will perform immunofluorescence staining on embryos that can develop normally and embryos that develop arrest, to obtain the role of histone modification in the development process of SCNT embryos.

[0104] 2. Test materials. Same as part of Test One.

[0105] 3. Test method.

[0106] Immunofluorescence staining. Collect cloned embryos at different stages of development, fix with 4% paraformaldehyde, permeabilize, block with blocking solution, and place the samples in H3K9me3 and γ-H2AX primary antibody solution, transfer to secondary antibody solution, and finally stain with Hoechst 33342, mount after mounting, and perform microscopy.

[0107] 4. Results.

[0108] 4.1 H3K9me3 in cloned embryos and in vitro fertilized embryos.

[0109] Differences in H3K9me3 in cloned embryos and in vitro fertilized embryos, such as Figure 8 H3K9me3 immunofluorescence in in vitro fertilized embryos and in SCNT embryos at different developmental stages, Figure 8 Figure 3: H3K9me3 fluorescence in in vitro fertilized embryos (A) and in SCNT embryos (B) (400x); C: Relative integrated optical density of H3K9me3 in in vitro fertilized embryos; D: Relative integrated optical density of H3K9me3 in SCNT embryos; E: Relative difference of H3K9me3 in in vitro fertilized embryos and in SCNT embryos.

[0110] In order to explore the expression pattern of H3K9me3 in SCNT embryos and in vitro fertilized embryos, immunofluorescence staining was performed on 1-cell, 2-cell and 4-cell in vitro fertilized embryos and SCNT embryos. It was found that the histone modification H3K9me3 was gradually down-regulated in in vitro fertilized embryos from 1-cell to 4-cell development, and was down-regulated in SCNT embryos from 1-cell to 2-cell development, but was up-regulated at 4-cell; in 1-cell to 4-cell embryos, especially at 4-cell, the level of H3K9me3 modification in SCNT embryos was significantly higher than that in in vitro fertilized embryos.

[0111] 4.2 H3K9me3 modification pattern in development-arrested and normally developed cloned embryos.

[0112] Differences in H3K9me3 in development-arrested and normally developed cloned embryos, such as Figure 9 H3K9me3 immunofluorescence in development-arrested and normally developed embryos, Figure 9 Figure 4: H3K9me3 fluorescence in development-arrested (A) and normally developed (B) SCNT embryos (400x); C: Relative integrated optical density of H3K9me3 in development-arrested and normally developed SCNT embryos.

[0113] In order to explore the differences in H3K9me3 in development-arrested and normally developed SCNT embryos, immunofluorescence staining was performed on development-arrested and normally developed 4-cell SCNT embryos, respectively, and it was found that the level of H3K9me3 modification in 2-cell arrested embryos was significantly higher than that in embryos developed to 4-cell. This phenomenon suggests that in SCNT embryos, the original silent epigenetic memory (such as H3K9me3) of the donor cell has not been effectively eliminated, resulting in arrest at the 2-cell stage.

[0114] 4.3 Differences in γ-H2AX in development-arrested and normally developed cloned embryos and in vitro fertilized embryos, such asFigure 10 Developmental arrested embryos and normally developing embryos γ-H2AX immunofluorescence, Figure 10 Figure A is the fluorescence state of γ-H2AX in developmentally arrested and normally developing fertilized embryos (400x); Figure B is the fluorescence state of γ-H2AX in developmentally arrested and normally developing SCNT embryos (400x); Figure C is the relative integrated optical density of γ-H2AX in fertilized embryos; Figure D is the relative integrated optical density of γ-H2AX in SCNT embryos.

[0115] In order to explore the expression differences of γ-H2AX in developmentally arrested SCNT embryos and fertilized embryos and in normally developing SCNT embryos and fertilized embryos, immunofluorescence staining was performed on developmentally arrested and normally developing 4-cell fertilized embryos and SCNT embryos. The DNA damage detection results also showed that the 4-cell γ-H2AX modification level of SCNT embryos was significantly higher than that of in vitro fertilized embryos; in in vitro fertilized embryos and SCNT embryos, the γ-H2AX modification level of 2-cell arrested embryos was significantly higher than that of embryos developing to 4-cell, but the γ-H2AX modification level of 2-cell arrested SCNT embryos was lower than that of in vitro fertilized embryos.

[0116] 5. SCNT can reprogram differentiated somatic cells to a pluripotent state, but its development ability is poor. H3K9me3 is considered to be one of the markers of heterochromatin regions, and is an epigenetic obstacle to zygotic genome activation in SCNT embryos (Lachner et al. 2001). The results of the present application found that in in vitro fertilized embryos, H3K9me3 was gradually down-regulated from 1-cell to 4-cell stage, indicating that the silent marker was normally removed (Wang et al. 2018), creating a more open chromatin environment for embryo gene activation. In SCNT embryos, although H3K9me3 also experienced a removal process from 1-cell to 2-cell stage, it was up-regulated at 4-cell stage and the level was higher than that of IVF embryos, which indicated that the nucleus derived from somatic cells may have abnormalities in the reprogramming process and failed to fully remove the original silent memory, thereby affecting the normal development of SCNT embryos. In SCNT embryos, the H3K9me3 modification level of 2-cell arrested embryos was significantly higher than that of embryos developing to 4-cell, proving that H3K9me3 is one of the reasons for the development arrest of SCNT embryos.

[0117] γ-H2AX is a marker rapidly phosphorylated after DNA double-strand breaks, used to reflect intracellular DNA damage and its repair status (Kuo and Yang 2008). Our findings show that in SCNT embryos, the γ-H2AX modification level in 2-cell arrested embryos was significantly higher than in 4-cell embryos. Simultaneously, the γ-H2AX level in 4-cell stage SCNT embryos was significantly lower than in developmentally arrested SCNT embryos, possibly because during SCNT, the differentiation memory of donor cells and incomplete epigenetic reprogramming often induce additional replication stress and DNA damage. Therefore, the higher γ-H2AX level in 4-cell stage SCNT embryos suggests that these embryos experience more DNA double-strand breaks or other forms of DNA damage during early cell division. Researchers have demonstrated a positive correlation between developmental impairment in SCNT embryos and the degree of DNA damage (Bohrere et al. 2014). Appropriate use of HDAC inhibitors not only helps repair DNA damage caused by SCNT but also improves embryonic developmental quality, providing an effective strategy for improving cloning efficiency.

[0118] 6. The H3K9me3 modification level in in vitro fertilized embryos showed a gradual downward trend from 1-cell to 4-cell; in SCNT embryos, H3K9me3 showed a decreasing trend from 1-cell to 2-cell stages, but an upward trend in 4-cell stage; overall, especially in the 4-cell stage, the H3K9me3 level in SCNT embryos was higher than that in in vitro fertilized embryos; at the same time, in SCNT embryos, the H3K9me3 level in 2-cell arrested embryos was significantly higher than that in embryos that developed to 4-cell stage. P <0.05).

[0119] As an indicator of DNA damage, γ-H2AX modification levels in SCNT embryos were significantly higher than those in in vitro fertilized embryos at the 4-cell stage; γ-H2AX levels in 2-cell arrested in vitro fertilization and cloned embryos were significantly higher than those in embryos that had developed to the 4-cell stage. P <0.05).

[0120] Experiment 4: Effects of Setdb2 and Shprh on the development of cloned embryos.

[0121] 1. Setdb2 and Shprh play potential regulatory roles in SCNT embryonic development. To verify... Setdb2 and Shprh Regarding the role in SCNT embryos, this invention analyzes the effects of Setdb2siRNA or ShprhmRNA on SCNT embryo development by microinjection.

[0122] 2. Test materials. Same as part of test 1.

[0123] 3. Test method.

[0124] 3.1 Setdb2 siRNA design and synthesis.

[0125] Design siRNA of Setdb2, synthesized by Shanghai Biotech Co., Ltd., diluted with ddH2O to 20 μΜ.

[0126] Table 2 Interference fragment sequence

[0127] 3.2 Embryo microinjection. The SCNT embryos activated for 6 h were transferred to a microinjection liquid containing 150 μL, and a embryo fixation needle (inner diameter 20 μm) was installed, and Femtotip II microinjection needle (inner diameter 0.5 μm; Eppendorf) was connected. Inject mode completed injection, and the embryo was transferred to embryo culture solution for subsequent development evaluation.

[0128] 4. Results.

[0129] 4.1 Cloned embryo Setdb2 knockdown efficiency, as Figure 11 Setdb2 knockdown mRNA expression; The results showed that Setdb2-497 had no obvious knockdown effect on Setdb2 gene, and Setdb2-1170 and Setdb2-1597 had significant knockdown effect on Setdb2 gene (P<0.05), among which Setdb2-1597 had the best knockdown effect. The above experimental results showed that microinjection of interference fragments into SCNT embryos could effectively inhibit the expression of Setdb2 in SCNT embryos. P <0.05), among which Setdb2-1597 had the best knockdown effect. The above experimental results showed that microinjection of interference fragments into SCNT embryos could effectively inhibit the expression of Setdb2 in SCNT embryos.

[0130] 4.2 Effect of Setdb2 knockdown on cloned embryo development, Figure 12 SCNT embryo development after Setdb2 knockdown, Figure 12 Figure A shows the cleavage rate of SCNT embryos after injection of Setdb2 siRNA; Figure B shows the 4-cell development arrest rate of SCNT embryos after injection of Setdb2 siRNA; Figure C shows the development status of SCNT embryos.

[0131] After injection of Setdb2 siRNA, the cleavage rate results showed that the effect of injection of each siRNA on embryo cleavage was not significantly different (P>0.05) (Figure A), but compared with the control group, the 4-cell arrest of the experimental group injected with siRNA was significantly reduced (P<0.05) (Figure B). Figure 12 Figure 12 ​Figure 4 shows that injection of Setdb2 interfering fragments can effectively promote the development of SCNT embryos to 4-cell.

[0132] 4.3 Effect of Setdb2 knockdown on H3K9me3 of cloned embryos, Figure 13 Figure 4 shows that injection of Setdb2 interfering fragments can effectively promote the development of SCNT embryos to 4-cell. Figure 13 Figure 4 shows that injection of Setdb2 interfering fragments can effectively promote the development of SCNT embryos to 4-cell.

[0133] The results show that, compared with the control group, after injection of Setdb2 SiRNA, the fluorescence intensity of H3K9me3 significantly decreases, indicating that knockdown of Setdb2 can promote the development of SCNT embryos by reducing H3K9me3.

[0134] 4.4 Effect of overexpression of Shprh on the development of cloned embryos, Figure 14 Figure 4 shows that injection of Setdb2 interfering fragments can effectively promote the development of SCNT embryos to 4-cell. Figure 14 Figure 4 shows that injection of Setdb2 interfering fragments can effectively promote the development of SCNT embryos to 4-cell.

[0135] The results show that, compared with the control group, after injection of Setdb2 SiRNA, the fluorescence intensity of H3K9me3 significantly decreases, indicating that knockdown of Setdb2 can promote the development of SCNT embryos by reducing H3K9me3.

[0136] 4.5 Effect of overexpression of Shprh on DNA damage of cloned embryos, Figure 15 Figure 4 shows that injection of Setdb2 interfering fragments can effectively promote the development of SCNT embryos to 4-cell. Figure 15 Figure 4 shows that injection of Setdb2 interfering fragments can effectively promote the development of SCNT embryos to 4-cell.

[0137] The results show that, compared with the control group, after injection of Setdb2 SiRNA, the fluorescence intensity of H3K9me3 significantly decreases, indicating that knockdown of Setdb2 can promote the development of SCNT embryos by reducing H3K9me3.

[0138] 5. Setdb2 is a histone methyltransferase that mainly catalyzes the methylation modification of H3K9me3. By knocking down Setdb2 through siRNA, the level of H3K9me3 may be reduced, thereby improving the efficiency of nuclear reprogramming and promoting the development of SCNT embryos to 4-cell. Recent reports (Torrano et al. 2019) also confirm the experimental results of the present application.

[0139] Shprh is a multi-domain protein with E3 ubiquitin ligase activity, and its core functions include DNA damage repair (Lin et al. 2011, Motegi et al. 2006), promoting homologous recombination repair (Brühl et al. 2019), genome stability maintenance (Kang et al. 2019), etc. In the present application, by overexpressing Shprh, DNA damage repair is promoted, which is consistent with the viewpoint mentioned in the prior art. In addition, open chromatin can accelerate the process of DNA damage repair, making γ-H2AX dephosphorylated or cleared faster, so that the fluorescence intensity decreases. The present application shows that Shprh can promote SCNT embryo development by reducing DNA damage.

[0140] 6. Setdb2 is significantly up-regulated in the development-arrested SCNT embryo blastomere, and the results of Setdb2 knockdown show that siRNA-1597 significantly knocks down Setdb2, promoting the development of SCNT embryo 4-cell; after Setdb2 knockdown, H3K9me3 is significantly decreased compared with the control group, indicating that Setdb2 inhibits the development of SCNT embryo through histone modification, and knockdown promotes the totipotency conversion of SCNT embryo.

[0141] Shprh is significantly down-regulated in the development-arrested SCNT embryo, and the results of overexpression show that after Shprh overexpression, the development of SCNT embryo is significantly improved; at the same time, γ-H2AX is significantly down-regulated, indicating that Shprh can promote the development of SCNT embryo by reducing DNA damage.

[0142] From the above experiments, it can be seen that the present application builds a tracking system for SCNT embryo blastomere development fate by optimizing the small pore culture system of cloned embryos, blastomere separation technology and development tracking. Setdb2 is significantly up-regulated in the development-arrested SCNT embryo blastomere, and after knockdown, H3K9me3 is significantly decreased, promoting the development of SCNT embryo 4-cell. Shprh is significantly down-regulated in the development-arrested SCNT embryo, and after Shprh overexpression, γ-H2AX is significantly down-regulated, and the development of SCNT embryo 4-cell is significantly improved.

[0143] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principles of the present application, should be covered within the protection scope of the present application.

Claims

1. A gene that promotes the development of SCNT embryos, characterized in that, This gene is an RNA interference fragment resulting from Setdb2 knockdown, or it may be... Shprh Overexpressed genes.

2. The gene for promoting SCNT embryonic development according to claim 1, characterized in that, The RNA interference fragments after Setdb2 knockdown include: the knockdown site Setdb2-497, and the knockdown sequences are: SEQ ID NO: 9, SEQ ID NO:

10.

3. The gene for promoting SCNT embryonic development according to claim 1, characterized in that, The RNA interference fragments after Setdb2 knockdown include: the knockdown site Setdb2-1170, and the knockdown sequences are: SEQ ID NO: 11, SEQ ID NO:

12.

4. The gene for promoting SCNT embryonic development according to claim 1, characterized in that, The RNA interference fragments after Setdb2 knockdown include: the knockdown site Setdb2-1597, and the knockdown sequences are: SEQ ID NO: 13, SEQ ID NO:

14.

5. The gene for promoting SCNT embryonic development according to claim 1, characterized in that, Shprh The overexpressed gene is SEQ ID NO: 1; used for γ-H2AX downregulation.

6. A system for tracking the developmental fate of scintillator blastomeres in SCNT embryos for screening genes that promote SCNT embryo development as described in claim 1, characterized in that, This system was built by optimizing the clonal embryo microwell culture system, blastomere separation technology, and developmental tracking. Based on this SCNT embryo blastomere development fate tracking system, combined with single-cell transcriptome sequencing, genes related to SCNT embryo development that promote SCNT embryo development were discovered and screened.

7. The system for tracking the developmental fate of scintillator blastomeres in SCNT embryos according to claim 6, characterized in that, The aim was to identify and screen genes associated with SCNT embryo development that promote SCNT embryo development, specifically RNA interference fragments after Setdb2 knockdown, or... Shprh Overexpressed genes.

8. A method for constructing a system for tracking the developmental fate of blastomeres in SCNT embryos, characterized in that, The method for constructing the SCNT embryo blastomere development fate tracking system as described in claim 6 includes: constructing the SCNT embryo blastomere development fate tracking system by optimizing the cloned embryo microwell culture system, blastomere separation technology and development tracking; The construction of the system for tracking the developmental fate of blastomeres in SCNT embryos specifically includes: S1 involves oocyte collection and maturation culture, primary cell culture, cell passage, cryopreservation and thawing, somatic cell nuclear transfer, sperm preparation, and in vitro fertilization. S2, then a small-well culture system is used to separate the blastomeres of the cloned embryos; S3, Statistical Analysis: SPSS software was used for one-way ANOVA to analyze the development of cloned embryos; ImageJ analysis was used for relative integrated optical density; and 2-1 analysis was used for gene expression. −ΔΔCT Analysis by means; experimental results are expressed as mean ± standard error.

9. The method for constructing a system for tracking the developmental fate of SCNT embryo blastomeres according to claim 7, characterized in that, Step S2, the microplate culture system includes: selecting cloned embryos with uniformly divided blastomeres, placing them in proteinase K solution, separating the blastomeres with a needle, washing one with PBS and transferring it into a PE tube for cryopreservation, and transferring the other homologous blastomer into the wells of the microplate and labeling it.

10. The method for constructing a system for tracking the developmental fate of scintillator embryos according to claim 8, characterized in that, Step S2: In the micro-well culture system, the well spacing is 500 µm; Step S2, blastomere separation of cloned embryos, SCNT embryo blastomere separation time 30 h.

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