Method for improving developmental potential of cloned embryos through high expression of Smarca5 and application

Abstract

The invention discloses a method for improving developmental potential of cloned embryos through high expression of Smarca5 and application of the method. According to the method, after the SCNT embryo is reconstructed, the proportion of 4 cell periods, the blastocyst formation rate and the proportion of inner cell clusters are remarkably increased by microinjection of Smarca5mRNA (500ng / mL). Immunofluorescence and qPCR results show that the expression of EGA related genes such as Polr2a and Dppa2 is up-regulated through high expression of the Smarca5, the synthesis level of new RNA is enhanced, and the abundance of H3K9me3 is remarkably reduced by adjusting the balance of Kdm4b / Suv39h2. According to the method, the embryo epigenetic reprogramming level is improved, the SCNT development quality is remarkably improved, and the method can be widely applied to the fields of animal cloning and embryo engineering.

Description

Technical Field

[0001] The present invention relates to a method for promoting somatic cell nuclear transfer (SCNT) embryo development by regulating Smarca5, particularly by enhancing embryonic genome activation (EGA) to improve the quality and efficiency of cloned embryo development. Specifically, the present invention relates to a method and application for enhancing the developmental potential of cloned embryos by overexpressing Smarca5. The present invention belongs to the field of animal embryo technology. Background Art

[0002] Somatic cell nuclear transfer (SCNT) is an important cloning technology that achieves embryonic development by transplanting somatic cell nuclei into enucleated oocytes. Because the mechanism of donor cell reprogramming is still unclear, the development rate and transplantation pregnancy rate of cloned embryos are still relatively low, which seriously hinders its practical application in animal breeding. Studies have found that the development efficiency of SCNT embryos is low, mainly due to insufficient regulation of gene expression during embryonic genome activation (EGA). During the SCNT process, after somatic cells are injected into enucleated oocytes, chromatin remodeling factors are needed to regulate histone modifications, so that the chromatin changes from a condensed state to an open state. The open chromatin enables transcription factors to efficiently bind to DNA and transcribe, thereby allowing a terminally differentiated somatic cell to regain totipotency.

[0003] Embryonic genome activation (EGA) is a critical step in the development of somatic cell cloned embryos, a process during which the genome gradually transitions from a silent state to an actively transcribed state. In recent years, numerous studies have revealed abnormalities in EGA in somatic cell cloned embryos and their underlying mechanisms. For example, H3K4me3 and H3K27me3 modifications are abnormally over-enriched in promoter regions of cloned embryos, leading to dysregulated gene expression. Abnormal enrichment of H3K9me3 modifications in cloned embryos is closely associated with transcriptional repression of genes involved in embryonic genome activation. Our group's research also found that reducing H3K9me3 levels can promote chromatin reprogramming and developmental competence in porcine somatic cell cloned embryos. Furthermore, abnormal chromatin remodeling in somatic cell cloned embryos also limits normal genome activation. For example, abnormalities in tertiary chromatin structure (topologically associated domains) lead to reduced chromatin accessibility, hindering the expression of genes involved in embryonic genome activation. Studies have shown that knocking out BRG1, a member of the ISWI family of chromatin remodelers, prevents transcription factor recruitment to nuclear DNA, leading to failure of embryonic genome activation. Furthermore, BRG1 regulates chromatin accessibility at OCT4 binding sites in embryonic stem cells, thereby promoting the transcription of pluripotency-related genes. Other studies have shown that the SWI / SNF complex maintains early embryonic totipotency by regulating the expression of pluripotency genes (such as OCT4 and NANOG), and its high expression accelerates the formation of the embryonic inner cell mass. This suggests that chromatin remodeling complexes play an important role in early embryonic development.

[0004] Smarca5 (SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5), a chromatin remodeling factor, has been shown to play a crucial role in embryonic development. It is the ATPase subunit of the ISWI family of chromatin remodeling complexes. Combining CRISPR activation (CRISPRa) with single-cell transcriptomics, Celia Alda-Catalinas et al. found that SMARCA5 plays a crucial role in regulating the transition from ESCs to 2-cell-like cells and genome activation. Recent studies have shown that SMARCA5 mediates the generation of aberrant chromatin accessibility in human leukemia stem cells. Chromatin reprogramming in cloned embryos is complex, and chromatin accessibility is often dysregulated, leading to failure of embryonic genome activation. SiRNA-mediated depletion of Smarca5 revealed that its absence leads to abnormal distribution of Oct4, reduced levels of Nanog and Sox17, reduced developmental competence in mouse embryos, and disrupted inner cell mass formation and differentiation. Loss of Smarca5 causes mouse embryos to arrest at an early stage, preventing them from completing normal development. Furthermore, Smarca5 is involved in regulating the self-renewal and differentiation of mouse embryonic stem cells and axial determination in Drosophila embryos. Knockout of Smarca5 leads to failure of MPF activation in mouse oocytes, resulting in meiotic arrest. Therefore, the functions of Smarca5 in different cell types or biological states require further elucidation. Existing research has largely focused on histone modifications, while limited research has examined the role of the chromatin remodeling factor Smarca5 in early mammalian embryonic development, particularly in embryonic genome activation.

[0005] Therefore, this study aims to analyze how Smarca5 affects embryonic genome activation and reveal the mechanism of Smarca5's role in the reprogramming of pig cloned embryos. This study will help to reveal the mechanism of reprogramming in somatic cell cloned embryos and provide theoretical support for explaining the root cause of low animal cloning efficiency. Summary of the Invention

[0006] The present invention aims to solve the problems of low pig SCNT embryo development efficiency and insufficient genome activation in the prior art, and provides a method for enhancing the developmental potential of cloned embryos by overexpressing Smarca5.

[0007] A method for improving the developmental potential of cloned embryos by overexpressing Smarca5. By microinjecting Smarca5 mRNA into enucleated oocytes, its overexpression is induced, thereby improving the efficiency of embryonic genome activation (EGA), the blastocyst formation rate and embryo quality. The results showed that the four-cell rate in the Smarca5 overexpression group was increased by 10.95% compared with the control group, with an increase of 20.9%; the blastocyst formation rate in the Smarca5 overexpression group was increased by 5.01% compared with the control group, with an increase of 28.2%.

[0008] Preferably, the Smarca5 mRNA concentration is 500 ng / mL, and the injection time is completed within 5 hours after SCNT.

[0009] Preferably, the high expression of Smarca5 can significantly increase the level of nascent RNA in four-cell stage SCNT embryos, and the nascent RNA level is detected by EU-488 incorporation staining.

[0010] Preferably, the nascent RNA level is detected by extracting total RNA and performing real-time fluorescence quantitative PCR, and the primers used are as follows:

[0011]

[0012]

[0013] Preferably, the high expression of Smarca5 can significantly upregulate the expression level of POLR2A protein and activate the mRNA expression of EGA key genes Dppa2 and Dnmt1.

[0014] Preferably, the high expression of Smarca5 can significantly increase the ratio of inner cell mass (ICM) to trophoblast (TE) cells, the total cell number of blastocyst and the blastocyst formation rate.

[0015] Preferably, the method can significantly improve the epigenetic state of porcine SCNT embryos at the EGA stage by:

[0016] (1) Downregulation of H3K9me3 abundance;

[0017] (2) Upregulation of demethylase Kdm4b expression;

[0018] (3) downregulating the expression of the methylase Suv39h2;

[0019] (4) Downregulate Dnmt1 expression, thereby reducing overall DNA methylation levels.

[0020] Preferably, the method does not change the cleavage rate, but significantly improves the efficiency of SCNT embryo development from the 4-cell stage to the blastocyst stage.

[0021] The present invention also protects an application of enhancing the developmental potential of cloned embryos through high expression of Smarca5.

[0022] Preferably, the application is used to promote genome activation, improve blastocyst quality, and improve somatic cell cloning efficiency in livestock breeding technology.

[0023] The beneficial effects of the present invention are:

[0024] 1. The high expression of Smarca5 significantly improves the developmental potential of somatic cell nuclear transfer embryos, specifically by increasing the proportion of 4-cell stage embryos, improving the blastocyst formation rate, and optimizing the blastocyst quality by increasing the total cell number and inner cell mass ratio.

[0025] 2. Enhance the embryonic genome activation markers, significantly upregulate the synthesis level of new RNA and the expression of the key EGA gene Dppa2, providing key transcriptional support for the early development of cloned embryos.

[0026] 3. Optimize epigenetic reprogramming of SCNT embryos by reducing the abundance of the repressive histone mark H3K9me3 and enhancing chromatin openness, thereby improving the epigenetic state of cloned embryos and improving reprogramming efficiency.

[0027] 4. Compared with traditional cloning techniques, this method is simple to operate, requiring only microinjection of Smarca5 mRNA (500 ng / mL) within 5 hours of SCNT reconstruction. It does not require complex gene editing or additional culture steps and is compatible with existing technology systems. It significantly increases the proportion of four-cell to blastocyst embryos while maintaining the embryonic cleavage rate, significantly improving embryo development efficiency and quality. This method can be widely used in somatic cell cloning of pigs, providing a new strategy for efficient and stable breeding of breeding stock. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the expression diagram of Smarca5 during the development of pig SCNT embryos according to the present invention.

[0029] Figure 1 (A) Western blot analysis of SMARCA5 protein expression in the metaphase of the second meiotic division of porcine oocytes, 1-cell, 2-cell, 4-cell, morula, and blastocyst of SCNT embryos.

[0030] Figure 1 (B) The relative expression levels of SMARCA5 protein in the metaphase of the second meiotic division of porcine oocytes, 1-cell, 2-cell, 4-cell, morula, and blastocyst of SCNT embryos were calculated using β-ACTIN as an internal reference.

[0031] Data are presented as the mean ± SEM of at least three independent experiments, n = 300. ***P < 0.001 compared with the control group.

[0032] Figure 2 This figure shows the effect of high expression of Smarca5 on the development of somatic cell nuclear transplanted embryos according to the present invention.

[0033] Figure 2 (A) Representative images of day 6 SCNT embryos from the control group and the Smarca5 overexpression group. Scale bar = 200 μm.

[0034] Figure 2 (B) Cleavage rates of SCNT embryos in the control group (n=331) and the Smarca5 overexpression group (n=332) at 48 hours.

[0035] Figure 2 (C) 4-cell cleavage rate of SCNT embryos in the control group (n=331) and the Smarca5 overexpression group (n=332).

[0036] Figure 2 (D) Blastocyst rate of SCNT embryos in the control group (n=255) and the Smarca5 high-expression group (n=257).

[0037] Figure 2 (E) Representative images of SMARCA5 immunofluorescence in blastocysts of the control group and the Smarca5 high-expression group, scale bar = 50 μm.

[0038] Figure 2 (F) Analysis of relative fluorescence intensity of SMARCA5 in blastocysts of the control group and the Smarca5 high-expression group, n = 10.

[0039] Figure 2 (G) Representative images of CDX2 immunofluorescence in blastocysts of the control group and the Smarca5 overexpression group, scale bar = 20 μm.

[0040] Figure 2 (H) Analysis of total cell number in blastocysts of the control group (n=15) and the Smarca5 high-expression group (n=16).

[0041] Figure 2 (I) Analysis of the ratio of inner cell mass to trophoblast in the control group (n=15) and the Smarca5 high-expression group (n=16).

[0042] The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group.

[0043] Figure 3 This figure shows the effect of high expression of Smarca5 on genome activation of somatic cell nuclear transfer embryos according to the present invention.

[0044] Figure 3(A) Representative images of EU-stained SCNT embryos at the 4-cell stage in the control group and the Smarca5 overexpression group. Scale bar = 50 μm.

[0045] Figure 3 (B) Analysis of EU relative fluorescence intensity at the 4-cell stage in SCNT embryos of the control group (n=18) and the Smarca5 overexpression group (n=22).

[0046] Figure 3 (C) RT-qPCR was used to detect the mRNA levels of EGA-related genes in SCNT embryo 4 cells of the control group (n=270) and the high-expression Smarca5 group (n=270).

[0047] Figure 3 (D) Western blotting was used to detect the protein expressions of SMARCA5 and POLR2A in SCNT embryo 4 cells of the control group and the Smarca5 high-expression group.

[0048] Figure 3 (E) β-Actin was used as an internal reference to calculate the relative expression levels of SMARCA5 and POLR2A proteins in the control group and the Smarca5 overexpression group at the 4-cell stage (n=270).

[0049] The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01 compared with the control group.

[0050] Figure 4 This figure shows the effect of high expression of Smarca5 on histone modification in the present invention.

[0051] Figure 4 (A) Representative images of H3K27ac immunofluorescence in the control group and the Smarca5 overexpression group. Scale bar = 20 μm.

[0052] Figure 4 (B) Analysis of relative immunofluorescence intensity of H3K27ac in the control group (n=14) and the Smarca5 overexpression group (n=13).

[0053] Figure 4 (C) Representative images of H3K9me3 immunofluorescence in the control group and the Smarca5 overexpression group. Scale bar = 20 μm.

[0054] Figure 4 (D) Analysis of relative immunofluorescence intensity of H3K9me3 in the control group (n=20) and the Smarca5 overexpression group (n=19).

[0055] Figure 4(E) RT-qPCR was used to detect the mRNA levels of genes related to H3K9me3 regulation in SCNT embryo 4 cells of the control group (n=270) and the high-expression Smarca5 group (n=270).

[0056] The data are presented as the mean ± SD of at least three independent experiments. *P < 0.05 compared with the control group.

[0057] Figure 5 This is an agarose gel electrophoresis diagram of the integrity detection of the in vitro transcription product Smarca5 mRNA of the present invention. DETAILED DESCRIPTION

[0058] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0059] A method and application for enhancing the developmental potential of cloned embryos through high expression of Smarca5.

[0060] Example 1 Materials and Methods

[0061] The pig ovaries and fetal tissues used were sourced from a designated slaughterhouse registered in Jilin Province. The donors were healthy Large White pigs. The sampling and processing procedures complied with the Jilin University Regulations on the Ethical Management of Experimental Animals (No. KT202402321) and were approved by ethics.

[0062] 1.1 Oocyte collection and in vitro maturation (IVM)

[0063] Fresh ovaries of Large White pigs were collected from a local slaughterhouse, placed in 34-37°C normal saline containing a double antibody, and brought back to the laboratory within 1 hour. Follicular fluid was extracted using a 10ml syringe with a No. 5 needle after rinsing the ovaries three times with preheated normal saline. The follicular fluid was cleaned three times with PBS containing 0.1% PVA. After completion, well-formed cumulus oocyte complexes (COCs) were picked up and placed in in vitro maturation culture medium (0.57mM TCM-199 (Gibco, BRL, Grand Island, NY, USA), 10% (v / v) porcine follicular fluid, 0.91mM sodium pyruvate, 10ng / mL epidermal growth factor, 100IU / mL follicle-stimulating hormone and luteinizing hormone). Subsequently, in a 38.5°C, 5% CO2, and saturated humidity carbon dioxide incubator, in vitro maturation culture was performed for 42-44 hours for subsequent experiments.

[0064] 1.2 Collection and culture of fetal fibroblasts

[0065] Isolate 30-35 day old porcine fetal skin fibroblasts (PEF). Use 75% alcohol to disinfect the fetus with intact fetal membranes, then wash with PBS solution, peel off the membranes, and take out the intact fetus. Remove the head, limbs, and internal organs of the fetus, wash the remaining tissue blocks with PBS and separate them into small pieces, place them in a clean culture dish and cut them into pieces less than 1mm2. Wash the cut tissue pieces with PBS, transfer them to a six-well plate, spread them out, turn them upside down and place them in an incubator at 37°C, 5% CO2 and appropriate humidity to culture for 1 hour, then add an appropriate amount of high-glucose DMEM culture medium containing 10% serum to allow fibroblasts to crawl out. The cell generation at this time is recorded as P0. When the cell density reaches 90%, it can be passaged or frozen. The cell generation used for somatic cell nuclear transplantation usually does not exceed P8.

[0066] 1.3 Somatic Cell Nuclear Transfer (SCNT)

[0067] After 44 hours of in vitro maturation of oocytes, cumulus cells were removed using 0.1% hyaluronic acid (HA). Oocytes with polar bodies were selected and placed in a paraffin oil droplet containing 5 μg / ml cytochalasin B. The oocyte nucleus was removed using blind aspiration under a micromanipulator. Appropriately sized porcine fetal fibroblasts were then selected and injected into the perivitelline space of the enucleated oocytes to form reconstructed embryos. Reconstructed embryos were fused using electrofusion using a NEPA GENE-ECFG21 instrument. The electrofusion parameters were 1.2 kV / cm, two unidirectional DC pulses of 60 μs each, with a 100 ms interval. The electrofusion medium was 300 mM mannitol buffer, and the procedure was performed at room temperature. Successfully fused somatic cell nuclear transfer embryos were cultured in PZM-5 culture medium at 38°C in an incubator with 5% CO2.

[0068] 1.4 Microinjection

[0069] SCNT embryos overexpressing Smarca5 were constructed using mRNA microinjection. Reconstructed embryos, after 5 hours of in vitro culture, were placed in a micromanipulator droplet covered with paraffin oil (containing 5 μg / ml cytochalasin B). Smarca5 mRNA (500 ng / ml) was injected using the micromanipulator. Successful injection was confirmed by the observation of a microscopic swell in the cytoplasm. After injection, the embryos were washed in PZM-5 and cultured in a 38°C, 5% CO2 incubator for another 6 days, designated as day 0. Blastocysts from the control and injected groups were collected on day 6 and Western blotting was performed to verify injection success.

[0070] 1.4.1 Preparation and purification of Smarca5 mRNA

[0071] The Smarca5 mRNA used in this study was obtained by in vitro transcription. The specific steps are as follows: First, the CDS region sequence of porcine Smarca5 was cloned and PCR amplified using Jima gene synthesis primers (the full length of the CDS region is 3159nt). The product was then ligated to the pGP-T7-MCS-polyA plasmid for amplification. The expression plasmid containing the Smarca5 open reading frame (ORF) was linearized and used as a template to synthesize mRNA using the T7 in vitro transcription system (MEGAscript T7 Kit, Thermo Fisher Scientific). After transcription, the product was capped (m7G Cap) and polyA tailed, and purified using the LiCl precipitation method to remove endotoxins and resuspended in RNase-free water. The integrity of the Smarca5 mRNA was tested as follows. Figure 5 As shown in the electrophoresis diagram, the A260 / 280 ratio is 2. After passing the test, the mRNA concentration is adjusted to 500 ng / mL and stored at -80°C for future use.

[0072] like Figure 5 The figure shows a gel electrophoresis of porcine Smarca5 mRNA produced by in vitro transcription. Three replicates (TY20132-1 to TY20132-3) are shown. The bands are clear and consistent in size, close to the expected 3159 nt, indicating that the mRNA has passed quality control. The band at the 1000 marker appears because single-stranded RNA easily forms secondary structures, resulting in multiple bands.

[0073] Microinjection was performed using a ZEISS-Vert.A1 micromanipulator with an Eppendorf micromanipulator arm, with an injection volume of approximately 10 μL per embryo. 180 SCNT embryos were injected into each experimental and control groups in three batches, with n = 3 biological replicates per group. After injection, the embryos were cultured in PZM-5 medium until the blastocyst stage.

[0074] 1.5 Immunofluorescence staining

[0075] SCNT embryos of the control group and the high expression group were collected at the four-cell stage (day 2) and blastocyst stage (day 6) and fixed in 4% paraformaldehyde at room temperature for 30 minutes.

[0076] After washing three times, permeabilize with 0.1% Triton-X100 for 30 minutes and wash again three times. The permeabilized SCNT embryos were blocked for 2 hours (PBS-PVA containing 1% BSA) and incubated with primary antibodies at 4°C overnight (Smarca5, 1:400; CDX2, 1:200; H3K9me3, 1:100; H3K27ac, 1:100). After incubation with the primary antibody, the embryos were washed three times in PBS-PVA and incubated in fluorescent secondary antibodies at room temperature for 1 hour. Finally, the sections were stained with 10mg / mL Hoechst33342 at room temperature for 10 minutes and then mounted. Finally, the number of trophoblast cells and blastocyst cells was counted and the protein fluorescence intensity was calculated using ImageJ software.

[0077] All immunofluorescence staining experiments included negative controls, using a nonspecific isotype antibody in place of the primary antibody to verify signal specificity. The optimized primary antibody concentration was determined through preliminary experiments, and all fluorescence images were acquired under the same microscopic parameters.

[0078] 1.6 Western blotting

[0079] SCNT embryos at the 4-cell stage, in vitro matured oocytes, parthenogenetically activated embryos, and somatic cell nuclear transfer embryos from the control and high-expression groups were collected at different developmental stages (1-cell stage, 13 hours; 2-cell stage, 24 hours; 4-cell stage, 48 hours; morula, 5 days; blastocyst, 6 days), with 90 embryos per group. These embryos were incubated in SDS lysis buffer (40% ddH2O, 50% glycerol, 10% SDS, 0.5 mM Tris-HCl, β-mercaptoethanol, and bromophenol blue) in a metal bath at 95°C for 10 minutes. Protein samples were separated on a 10% polyacrylamide gel containing 0.1% SDS and transferred to a PVDF membrane. The membrane was blocked with 5% BSA for 2 h at room temperature and incubated with the corresponding rabbit anti-SMARCA5 (1:2000, ab72499, Abcam), rabbit anti-POLR2A (1:1500, DF6831, Affinity Biosciences), and rabbit anti-β-ACTIN (1:1000, 20536-1-AP, Proteintech) antibodies at 4°C overnight. The membrane was then washed four times in TBST for 7 min each and incubated with secondary goat anti-rabbit IgG (1:5000, Bioworld Technology Inc, Louis Park, MN, USA, BS13278) for 1 h at room temperature. The blots were visualized using a Tanon 5200 image analyzer (Tanon, Shanghai, China) and analyzed using NIH ImageJ software.

[0080] 1.7EU staining

[0081] The levels of nascent RNA in embryos were detected using the EU-488 RNA synthesis detection kit (R0301S, Beyotime, Shanghai). First, 4-cell-stage SCNT embryos from the control and high-expression groups were transferred to PZM-5 medium containing 1 mM EU and incubated at 38.5°C and 5% CO2 for 3 hours. Then, the embryos were fixed in 4% paraformaldehyde for 30 minutes and permeabilized with 0.1% TritonX-100 for 1 hour at room temperature. After washing three times with PBS-PVA, the embryos were incubated with 500 μl of a color development solution (430 μl ClickReaction Buffer, 20 μl CuSO4 solution, 1 μl Azide 488 solution, 50 μl Click Additive Solution) at room temperature for 30 minutes in the dark. Next, the sections were stained with 10 mg / mL Hoechst33342 for 15 minutes and then mounted. Finally, the fluorescence intensity of 5-EU was analyzed using an inverted fluorescence microscope and ImageJ software.

[0082] 1.8 Total RNA extraction and real-time fluorescence quantitative PCR

[0083] Total RNA was extracted from four cells of 100 SCNTs in the control group and the high expression group using TRIzol (15596026CN, Invitrogen). Reverse transcription was performed using All-in-One 5X RT MasterMix (G592, abm) to obtain cDNA, and the cDNA was purified using Blastaq TM qPCR was performed using 2X qPCR MasterMix (G891, abm). Each 20 μl system contained BlasTaq TM 2X qPCR, cDNA, forward and reverse primers (10 μM), and nuclease-free water. The reaction program consisted of 40 cycles of three steps: 95°C for 3 min, 95°C for 15 s, and 60°C for 1 min. The 18S gene was used as an internal control, and 2 -ΔΔCT Methods Analysis Experimental Results The primer sequences used are shown in Table 1.

[0084] Table 1. Primer information used in RT-qPCR

[0085]

[0086] 1.9 Statistical analysis

[0087] Data are expressed as mean ± standard error of the mean (SEM). Each experiment used at least three independent biological replicates. All data were tested for normal distribution using GraphPad software. Significant differences among the four groups were analyzed using one-way analysis of variance (ANOVA). Significant differences between two independent groups were compared using the Student's t-test, with P < 0.05 indicating statistical significance. Graphs were drawn using GraphPad software.

[0088] Example 2 Analysis Results

[0089] 2.1 Dynamic expression of Smarca5 during porcine somatic cell nuclear transfer embryo development

[0090] This study first analyzed the expression pattern of SMARCA5 during the key stages of porcine SCNT embryo development. The results showed that SMARCA5 was expressed in mature oocytes and early SCNT embryos ( Figure 1 A). Compared with mature oocytes, SMARCA5 protein showed a significant downward trend during the key stages of SCNT embryo development, especially at the 4-cell stage, where its protein level dropped to the lowest point (P < 0.001), and then gradually recovered ( Figure 1 B) Given that EGA in early porcine embryos mainly occurs at the 4-cell stage, this result suggests that Smarca5 may play a crucial role in the EGA process of porcine nuclear transfer embryos.

[0091] 2.2 Effects of Smarca5 on the development of porcine somatic cell nuclear transfer embryos

[0092] To further explore the role of Smarca5 in SCNT embryo development, this study conducted a high-expression experiment of Smarca5 in SCNT embryos. The results showed that compared with the control group (80.53±3.240%), high-expression of Smarca5 had no effect on the cleavage rate at 48h (80.16±3.653, P>0.05). Figure 2 B), but significantly increased the percentage of four-cell stage ( Figure 2 C; control group: 52.34±1.196%, high expression group: 63.29±0.6433%, P<0.001), the four-cell rate in the Smarca5 high expression group increased by 10.95% compared with the control group, with an increase of 20.9%; and effectively promoted blastocyst formation ( Figure 2 A and 2D; control group: 17.77±0.9003%, high expression group: 22.78±0.9647%, P<0.01), the blastocyst formation rate in the Smarca5 high expression group was 5.01% higher than that in the control group, with an increase of 28.2%. Further analysis found that high expression of Smarca5 significantly increased the level of SMARCA5 protein in blastocysts ( Figure 2 E and 2F, P < 0.01). In addition, high expression of Smarca5 also significantly increased the total number of blastocyst cells ( Figure 2 G and 2H, P < 0.05) and the ratio of inner cell mass to trophoblast ( Figure 2 I, P < 0.01). These results indicate that high expression of Smarca5 can significantly promote the development of porcine somatic cell nuclear transfer embryos and effectively improve the quality of embryos.

[0093] In the above embodiment, the percentage of improvement in the high expression group relative to the control group was calculated using the following formula (including the four-cell and blastocyst improvement rates):

[0094] Improvement percentage = (percentage of high expression group - percentage of control group) / percentage of control group × 100%.

[0095] 2.3 Effect of Smarca5 on genome activation in porcine somatic cell nuclear transfer embryos

[0096] To investigate whether Smarca5 affects porcine somatic cell cloned embryonic genome activation (EGA), this study examined the levels of newly generated RNA in 4-cell SCNTs. The results showed that high expression of Smarca5 significantly increased the EU fluorescence intensity at the 4-cell stage ( Figure 3 A and 3B, P < 0.01). Further studies found that high expression of Smarca5 significantly increased the mRNA levels of genes related to EGA (Dppa2 and Dnmt1) ( Figure 3 C, P < 0.05 and P < 0.01). Protein detection results showed that after high expression of Smarca5, the level of RNA polymerase (POLR2A) was significantly increased ( Figure 3 D and 3E, P < 0.01). This indicates that high expression of Smarca5 can effectively promote genome activation in SCNT embryos. To further explore how Smarca5 affects EGA, this study examined the levels of histone modifications during EGA. The results showed that high expression of Smarca5 did not affect the abundance of activating histone H3K27ac ( Figure 4 A and 4B, P>0.05), but significantly reduced the abundance of H3K9me3, a chromatin condensation marker ( Figure 4 C and 4D, P < 0.05). The mRNA levels of genes involved in H3K9me3 methylation (Suv39h1, Suv39h2, and Setdb1) and demethylation (Kdm4a, Kdm4b, Kdm4c, and Kdm4d) were detected by qPCR. The results showed that high expression of Smarca5 significantly increased the mRNA level of the demethylation gene Kdm4b, while reducing the mRNA level of the methylation gene Suv39h2 ( Figure 4E, P < 0.05). These results indicate that overexpression of Smarca5 increases the level of Polr2a, thereby promoting the transcription of Kdm4b and Suv39h2, reducing the abundance of H3K9me3, thereby opening chromatin and activating EGA-related genes, ultimately promoting genome activation in nuclear transfer embryos.

[0097] In summary, during the embryonic genome activation (EGA) period (4-cell stage), the expression level of Smarca5 in porcine SCNT embryos was much lower than that in parthenogenetic activation (PA) embryos. Overexpression of Smarca5 significantly increased the rate of SCNT development to the 4-cell stage and the rate of blastocyst formation. In addition, the quality of SCNT embryos blastocysts was significantly improved. Compared with SCNT blastocysts, blastocysts with overexpression of Smarca5 had a larger total number of cells and a higher ratio of inner cell mass to trophoblast cells. Further studies found that overexpression of Smarca5 promoted EGA in SCNT embryos. Specifically, compared with control SCNT embryos, SCNT embryos in the overexpression group had higher levels of nascent RNA, protein levels of RNA polymerase (POLR2A) that regulates EGA, and mRNA levels of the EGA-related gene Dppa2. Most importantly, overexpression of Smarca5 can promote epigenetic reprogramming and enhance chromatin accessibility. Overexpression of Smarca5 significantly reduced the abundance of the chromatin condensation marker H3K9me3 by increasing the demethylase Kdm4b and reducing the mRNA level of the methylase Suv39h2. In addition, overexpression of Smarca5 reduced the overall DNA methylation level in SCNT embryos during the EGA period by reducing the mRNA level of DNA methyltransferase (Dnmt1). Therefore, this study provides a potential method for achieving higher cloning efficiency. Those skilled in the art can also reasonably expect that this method is also applicable to other mammals, such as cattle, sheep, and mice. These animals also have similar genome activation and epigenetic reprogramming processes during embryonic development, and Smarca5 also plays an important regulatory role in these species.

[0098] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0099] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for enhancing the developmental potential of cloned embryos by overexpressing Smarca5, characterized by: By microinjecting Smarca5 mRNA into enucleated oocytes during the reconstructed embryo stage, its high expression was induced, thereby improving the embryo's genomic activation efficiency, blastocyst formation rate and embryo quality. The results showed that the four-cell rate in the Smarca5 high-expression group was increased by 10.95% compared with the control group, with an increase of 20.9%; the blastocyst formation rate in the Smarca5 high-expression group was increased by 5.01% compared with the control group, with an increase of 28.2%.

2. The method of claim 1, wherein the method comprises: The concentration of Smarca5 mRNA was 500 ng / mL, and the injection time was completed within 5 hours after SCNT.

3. The method of claim 1, wherein the method comprises: The high expression of Smarca5 can significantly increase the level of nascent RNA in four-cell stage SCNT embryos, and the level of nascent RNA is detected by EU-488 incorporation staining.

4. The method of claim 3, wherein the method comprises: The levels of nascent RNA were detected by total RNA extraction and real-time fluorescence quantitative PCR. The primers used are as follows:

5. The method for enhancing the developmental potential of cloned embryos by overexpressing Smarca5 according to any one of claims 1 to 3, characterized in that: The high expression of Smarca5 can significantly upregulate the expression level of POLR2A protein and activate the mRNA expression of EGA key genes Dppa2 and Dnmt1.

6. The method for enhancing the developmental potential of cloned embryos by overexpressing Smarca5 according to any one of claims 1 to 3, characterized in that: The high expression of Smarca5 can significantly increase the ratio of inner cell mass (ICM) to trophoblast (TE) cells, the total number of blastocyst cells and the blastocyst formation rate.

7. The method of claim 1, wherein the method comprises: This method can significantly improve the epigenetic status of porcine SCNT embryos at the EGA stage by: (1) Downregulation of H3K9me3 abundance; (2) Upregulation of demethylase Kdm4b expression; (3) downregulating the expression of the methylase Suv39h2; (4) Downregulate Dnmt1 expression, thereby reducing overall DNA methylation levels.

8. The method of claim 1, wherein the method comprises: This method does not change the cleavage rate, but significantly improves the efficiency of SCNT embryo development from the 4-cell stage to the blastocyst stage.

9. An application of enhancing the developmental potential of cloned embryos by overexpressing Smarca5.

10. The use of claim 9 for enhancing the developmental potential of cloned embryos by overexpressing Smarca5, characterized in that: This application is used to promote genome activation, improve blastocyst quality, and increase somatic cell cloning efficiency in livestock breeding technology.

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