Method for improving developmental capacity of pig somatic cell nuclear transfer embryo and application

Abstract

The invention discloses a method for improving the developmental capacity of a pig somatic cell nuclear transfer embryo and application, and belongs to the technical field of embryo culture. The invention aims to improve the success rate of pig somatic cell nucleus embryo transfer. The invention provides a method for improving the ex-embryonic developmental capacity of pig somatic cell nuclear transfer, which comprises the following steps: adding a small molecule inhibitor into a somatic cell culture solution used for somatic cell nuclear transfer to reduce somatic cell H3K9me3 modification, and carrying out somatic cell nuclear transfer by using PEF with reduced H3K9me3 modification as donor cells to improve the somatic cell nuclear transfer developmental capacity. The cell culture solution is prepared from the following components: a DMEM (Dulbecco Modified Eagle Medium) culture medium is used as a basic culture solution, and 15% fetal calf serum, OTS186935, F5446 and SETDB1-TTD-IN-1 are added into the basic culture medium. The culture solution can reduce H3K9me3 modification of donor cell PEF and improve the in-vitro developmental capacity of pig somatic cell nuclear transfer embryos.

Description

Technical Field

[0001] The invention belongs to the technical field of embryo culture, and in particular relates to a method for improving the developmental ability of pig somatic cell nuclear transplanted embryos and its application. Background Art

[0002] The production of cloned pigs via somatic cell nuclear transfer (SCNT) holds great promise for applications in animal husbandry and biomedicine. Despite significant efforts over the past two decades, such as modulating embryonic epigenetic modifications and knocking out XIST in donor cells, the birth rate of full-term cloned pigs remains low.

[0003] Notably, SCNT embryos still exhibit developmental impairments during the maternal-to-zygotic genome activation (ZGA) period, which is believed to be caused by incomplete epigenetic reprogramming of the somatic cell genome. H3K9me3-mediated heterochromatin is also considered a barrier to cell fate changes. However, siRNA and mRNA synthesis are economically expensive, unstable, easily degraded, and exhibit dosage effects. Furthermore, injection requires high technical skills, is inefficient, and can damage the embryo. These technical bottlenecks severely restrict the industrial application of cloning technology. Summary of the Invention

[0004] The purpose of the present invention is to improve the success rate of porcine somatic cell nuclear transfer embryos while reducing embryo damage. A method has been developed to treat donor cells with small molecule inhibitors to reduce the H3K9me3 level of donor cells, use the treated cells for somatic cell nuclear transfer, improve the abnormal zygotic genome activation (ZGA) in the early developmental stage of somatic cell nuclear transfer embryos, improve the reprogramming efficiency, and ultimately significantly improve the in vitro development efficiency of porcine somatic cell nuclear transfer embryos.

[0005] The invention provides a porcine PEF somatic cell culture fluid. The cell culture fluid consists of the following components: a DMEM culture medium as a basic culture fluid, to which 15% fetal bovine serum, OTS186935, F5446 and SETDB1-TTD-IN-1 are added.

[0006] The invention provides a porcine PEF somatic cell culture fluid, which consists of the following components: a DMEM culture medium as a basic culture fluid, to which 15% fetal bovine serum, 1 μM OTS186935, 0.25 μM F5446 and 1 μM SETDB1-TTD-IN-1 are added.

[0007] The invention provides a porcine PEF somatic cell culture fluid, which is composed of the following components: a DMEM culture medium as a basic culture fluid, 15% fetal bovine serum, OTS186935, F5446 and SETDB1-TTD-IN-1 added to the basic culture fluid at a mass concentration of 4:1:4.

[0008] The present invention provides a method for improving the in vitro development ability of pig somatic cell nuclear transplanted embryos. The method comprises culturing pig PEF somatic cells in the above-mentioned culture medium for 3-6 days and then performing nuclear transplantation.

[0009] Further defined, the culture conditions were 5% CO2 and 95% air at 37°C.

[0010] The present invention provides a method for preparing the above-mentioned cell culture fluid, and the steps of the method are as follows: taking 42.5ml of DMEM basic culture fluid, adding 7.5ml of serum, adding OTS186935, F5446 and SETDB1-TTD-IN-1, mixing, and filtering the liquid with a 0.22um sterile filter.

[0011] It is further defined that the mass concentration of OTS186935 is 1 μM, the mass concentration of F5446 is 0.25 μM, and the mass concentration of SETDB1-TTD-IN-1 is 1 μM.

[0012] It is further defined that the mass concentration ratio of OTS186935, F5446 and SETDB1-TTD-IN-1 is 4:1:4.

[0013] The present invention provides an application of the above-mentioned culture solution in improving the in vitro development ability of pig somatic cell nuclear transplanted embryos.

[0014] It is further defined that after reducing the H3K9me3 modification of pig PEF somatic cells, somatic cell nuclear transplantation is performed to improve the in vitro development of somatic cell nuclear transplantation.

[0015] Beneficial Effects: OTS186935 is a potent inhibitor of the H3K9me3 histone methyltransferase SUV39H2, F5446 is a selective small molecule inhibitor of the SUV39H1 methyltransferase, and SETDB1-TTD-IN-1 can increase SETDB1-TTD-IN-1 methyltransferase activity in cells. When used together, these three agents effectively reduce H3K9me3 modification in donor PEF cells. Furthermore, using H3K9me3-reduced PEFs as donor cells for somatic cell nuclear transfer (SCNT) has been shown to improve the in vitro developmental capacity of porcine SCNT embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The figure shows the results of H3K9me3 immunofluorescence after culturing PEF cells with OTS186935, F5446, and SETDB1-TTD-IN-1 for 3 days;

[0017] Figure 2 This is the result of H3K9me3 immunofluorescence at the 4C stage of nuclear transplantation after the H3K9me3 modification of PEF was reduced by OTS186935, F5446 and SETDB1-TTD-IN-1 inhibitors;

[0018] Figure 3 This is the result of H3K9me3 immunofluorescence at the 8C stage of nuclear transplantation after the H3K9me3 modification of PEF was reduced by OTS186935, F5446 and SETDB1-TTD-IN-1 inhibitors;

[0019] Figure 4 The results of principal component analysis of SCNT embryos in the control group and experimental group are shown;

[0020] Figure 5 This figure shows the developmental results of somatic cell nuclear transfer embryos after the H3K9me3 modification of PEF was reduced using OTS186935, F5446 and SETDB1-TTD-IN-1 inhibitors. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the embodiments, and the advantages of the present invention will become clearer as the description progresses. It should be understood that the scope of protection claimed by the present invention is not limited by the specific embodiments described. The specific embodiments provided by the present invention are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art can modify the specific embodiments of the present invention or make equivalent replacements for some technical features with reference to the description of the specification. Such improvements and replacements that do not require creative work should also fall within the scope of protection of the claims attached to the present invention.

[0022] Reagent sources: All experimental reagents in the present invention were purchased from Sigma (USA), except for those otherwise specified: rabbit anti-H3K9me3 antibody (AB8898), secondary antibodies (Invitrogen, A21207 and A11008), DMEM (12100-046), SingleCell Full Length mRNA-Amplification Kit (N712) (vazyme), TruePrep DNA Library Prep Kit V2 (TD503) (vazyme) and Index Kit V2 (TD202) (vazyme).

[0023] OTS186935 (MCE, HY-122181), F5446 (MCE, HY-150190) and SETDB1-TTD-IN-1 (MCE, HY-141539)

[0024] Example 1: Preparation of donor cells for somatic cell nuclear transfer

[0025] P1 porcine embryonic fibroblasts (PEF) were revived from liquid nitrogen and seeded in DMEM supplemented with 15% fetal bovine serum (FBS) at 37°C and 5% CO2 for primary expansion. After three days of adherent growth and reaching approximately 90% confluence, the cells were digested and passaged using 0.25% trypsin-EDTA. The culture medium was treated with the inhibitory system OTS186935 (1 μM), F5446 (0.25 μM), and SETDB1-TTD-IN-1 (1 μM) for 72 hours before treatment. A control group received no inhibitors. Some cells were cryopreserved in a 10% DMSO-containing cryopreservation buffer using a programmed temperature-lowering procedure. Other cells were fixed with 4% paraformaldehyde for 30 minutes, washed three times with PBS, and permeabilized with 0.1% Triton X-100 before immunofluorescence staining of target proteins. A control group without adding inhibitors was set up at the same time as the subculture.

[0026] Example 2: Preparation of pig nuclear transfer embryos

[0027] Step 1: The pig ovaries collected from the slaughterhouse were transported back to the laboratory in 37°C physiological saline, and 2-8mm follicles were extracted to obtain cumulus oocyte-oocyte complexes (COCs). High-quality COCs were selected by three gradient sedimentation washings with Hepes buffer, and cultured in vitro for 42-44 hours at 38.5°C and 5% CO2 containing maturation culture medium; then, mature oocytes were separated by digestion with 0.1% hyaluronidase at 37°C for 3 minutes, and the individuals with released first polar bodies were selected as nuclear transfer recipients; after enucleation by micromanipulation, porcine embryonic fibroblasts (PEFs) were injected into the perivitelline space, and reconstructed embryos were constructed by 120V / mm double-pulse electrofusion, and finally, in vitro culture was completed in PZM-3 medium at 39°C and 5% CO2.

[0028] Step 2: Oocyte maturation in vitro

[0029] Porcine ovaries were collected from the slaughterhouse and transported to the laboratory within 1 hour of being placed in 37°C saline. After the ovaries were temperature-checked, they were gently drained with a strainer and then rinsed with 37°C saline supplemented with double-antibody antibodies. COCs were extracted from follicles 2 to 8 mm in diameter using a 10mL syringe with a 10G needle. The extracted follicular fluid was placed in a 50mL conical-bottom centrifuge tube in a 37°C water bath. After oocyte retrieval, the tube was placed in an incubator and allowed to settle for 10 minutes. The supernatant follicular fluid was discarded, and the tube was washed with HEPES. The tube was shaken and allowed to settle for 3 minutes in an incubator. The supernatant was discarded. This wash was repeated twice, and HEPES was added for the third time before the tube was placed in a large domestic dish for egg collection. COCs containing intact cumulus cells and homogeneous cytoplasm were selected at 1x magnification. The resulting COCs were then cultured for in vitro maturation. The selected COCs were cultured in a mature culture medium at 38.5°C, 5% CO2, 95% air, and saturated humidity for 42 to 44 hours.

[0030] Step 3: Retrieval of mature oocytes

[0031] After culturing the COCs in step 2 for 42 to 44 hours, transfer approximately 200 COCs into a 700 μl tube of 0.1% hyaluronidase (preheated in a 37°C incubator) and vortex for 3 minutes. After vortexing, transfer the permeabilization enzyme into the operating solution to terminate digestion. Detect the oocytes as quickly as possible and place them in another small dish containing more operating solution. Under a microscope, use the release of the first polar body as the criterion for oocyte maturity and select mature oocytes.

[0032] Step 4: Somatic cell nuclear transfer and in vitro culture of transplanted embryos

[0033] The oocytes selected in step 3 with distinct polar bodies and uniform cytoplasm were selected as recipients. The oocyte nucleus was removed by aspiration of the first polar body and adjacent cytoplasm through a glass tube. A single PEF (not treated with small molecule inhibitors) was then injected into the perivitelline space of the oocyte using an injection needle. The reconstructed embryos were then fused by electroporation using two direct pulses of 120 V / mm for 30 ms in the fusion medium. The reconstructed embryos were cultured in PZM-3 medium at 39°C in an atmosphere of 5% CO2 and 95% air.

[0034] Example 3: Using OTS186935, F5446, and SETDB1-TTD-IN-1 inhibitors to reduce the efficiency of H3K9me3-modified somatic cell nuclear transfer embryos in PEFs

[0035] PEF cells treated with OTS186935, F5446, and SETDB1-TTD-IN-1 in Example 1 were selected for nuclear transplantation, and the detailed steps were carried out with reference to Example 2. Nuclear-transferred embryos were cultured in PZM-3 and placed in an incubator at 38.5°C, 5% CO2, and saturated humidity. ZGA samples were obtained after 84 hours of culture, and blastocyst samples were obtained after 6 days of culture.

[0036] Comparative Example 1:

[0037] SCNT embryos from Example 2 (the donor PEF cells used in this somatic cell nuclear transfer were not treated with small molecule inhibitors) were cultured in PZM-3 culture medium at all stages in an incubator at 38.5°C, 5% CO2, and saturated humidity. ZGA samples were obtained after 84 hours of culture, and blastocyst samples (control group PZM-3) were obtained after 6 days of culture.

[0038] Effect experiment:

[0039] (1) Immunofluorescence detection

[0040] Cell samples obtained in Example 1, Example 2, and Comparative Example 1, and ZGA stage or blastocyst samples were placed in a 96-well plate, fixed with 4% paraformaldehyde solution, permeabilized with 1% Triton X-100 in PBS, and fixed with 1% BSA in PBS for 1 hour. Immunolabeling: Embryos were incubated overnight at 4°C, washed three times with a primary antibody against H3K9me3, and incubated with a secondary antibody for 1 hour, diluted 1:2000 in fixing solution. Samples were washed and stained with 5 mg / mL Hoechst 33342. Fluorescence was detected and imaged using a Nikon fluorescence microscope.

[0041] (2) Transcriptome sequencing of somatic cell nuclear transfer embryos

[0042] The blastocysts of Example 3 and Comparative Example 1 were subjected to single-cell transcriptome sequencing analysis using SMART-seq technology. The specific process included: using Vazyme's Single Cell Full Length mRNA-Amplification Kit (N712) to complete embryonic zona pellucida removal (1% HCl acidic MAN solution), lysis and cDNA amplification, and generating full-length cDNA by reverse transcription (42°C for 90 minutes) and 15 rounds of PCR amplification (98°C denaturation / 65°C annealing / 72°C extension); then using TruePrepDNALibrary Prep Kit V2 (TD503) and Index Kit V2 (TD202) for library construction, after 55°C TTE Mix fragmentation, 15-cycle PCR enrichment and VAHTS magnetic bead sorting, the library quality control was completed by Agilent 2100 and Qubit 4. Sequencing was performed on the IllμMina platform. The raw data were quality-controlled by FastQC and then aligned to the EnsemblSus scrofa 11.1 genome (version 105) using Hisat2 / Bowtie2. Gene expression was analyzed by Stringtie (TPM quantification) or Subread (Reads Count statistics). Default parameters were used throughout the process to ensure consistency of the results.

[0043] (3) Effects of somatic cell nuclear transfer on embryonic development in vitro:

[0044] The blastocyst rates (number of blastocysts / total number of nuclear transplants×100%) after 6.5 days of culture in Example 3 and Comparative Example 1 were statistically analyzed.

[0045] result:

[0046] 1) Effect of OTS186935+F5446+SETDB1-TTD-IN-1 on H3K9me3 modification in porcine fetal fibroblasts

[0047] After PEF cells were treated with OTS186935, F5446 and SETDB1-TTD-IN-1 for 3 days, the H3K9me3 modification levels in the experimental group and the control group were detected. We found that only when the three inhibitors were combined could the H3K9me3 modification level of PEF cells be significantly reduced ( Figure 1 ).

[0048] 2) H3K9me3 modification in porcine somatic cell nuclear transfer embryos at the ZGA stage in vitro treated with OTS186935, F5446, and SETDB1-TTD-IN-1 in PEF cells:

[0049] Somatic cell nuclear transfer was performed using PEFs with reduced H3K9me3 modification, and samples were collected at 4-8C for H3K9me3 IF staining. The results showed that the H3K9me3 histone modification levels of SCNT-4C and SCNT-8C embryos obtained in the experimental group were significantly reduced ( Figure 2 and Figure 3 ).

[0050] 3) Effects of combined treatment of PEF cells with OTS186935, F5446, and SETDB1-TTD-IN-1 on embryonic transcription following SCNT:

[0051] The results of principal component analysis showed that the transcriptome data of SCNT embryos obtained by treating donor PEF cells with OTS186935, F5446 and SETDB1-TTD-IN-1 and then performing somatic cell nuclear transplantation were different from the transcriptome characteristics of SCNT embryos in the control group. This result indicates that the combined treatment of OTS186935, F5446 and SETDB1-TTD-IN-1 can significantly improve the transcriptional reprogramming efficiency of porcine SCNT embryos. Figure 4 This optimization of transcriptome characteristics may be related to the regulation of H3K9me3 modification levels, suggesting that combined treatment with OTS186935, F5446, and SETDB1-TTD-IN-1 partially repairs transcriptional abnormalities present in SCNT embryos during reprogramming through the regulation of epigenetic modifications, thereby improving the developmental potential of the embryos.

[0052] 4) Developmental status of PEF cells after nuclear transfer following treatment with OTS186935, F5446, and SETDB1-TTD-IN-1:

[0053] SCNT was performed using PEF cells that had H3K9me3 deleted, and the blastocyst rate was counted until D6.5. The results showed no significant difference in cleavage rate between the experimental and control groups, but the blastocyst rate in the experimental group increased by 8.9%.

[0054] Table 1. In vitro development statistics of SCNT embryos using PEF cells that erased H3K9me3 modification

[0055] Group Number of nuclear transplants Cleavage (%) Blastocyst (%) control group 117 13 11.1 Treatment group 125 25 20

[0056] In summary, by adding small molecule inhibitors to reduce the H3K9me3 modification of donor cell PEF and using the treated PEF as donor cells for somatic cell nuclear transplantation, the efficiency of somatic cell nuclear transfer can be improved, the blastocyst rate can be increased, and the efficiency of reprogramming can be improved.

[0057] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A porcine fetal fibroblast culture medium, characterized in that: The cell culture fluid consists of the following components: DMEM culture medium is used as the basic culture fluid, 15% fetal bovine serum, OTS186935, F5446 and SETDB1-TTD-IN-1 are added to the basic culture fluid.

2. A porcine fetal fibroblast culture medium, characterized in that: The cell culture fluid consists of the following components: DMEM culture medium is used as the basic culture fluid, and 15% fetal bovine serum, 1 μM OTS186935, 0.25 μM F5446 and 1 μM SETDB1-TTD-IN-1 are added to the basic culture fluid.

3. A porcine fetal fibroblast culture medium, characterized in that: The cell culture fluid consists of the following components: DMEM culture medium is used as the basic culture fluid, 15% fetal bovine serum, OTS186935, F5446 and SETDB1-TTD-IN-1 are added to the basic culture fluid at a mass concentration of 4:1:

4.

4. A method for improving the in vitro developmental ability of porcine somatic cell nuclear transfer embryos, characterized in that: The porcine fetal fibroblasts are cultured in the culture medium according to any one of claims 1 to 3 for 3 to 6 days, and then nuclear transplantation is performed.

5. The method according to claim 4, characterized in that The culture conditions were 5% CO2 and 95% air at 37°C.

6. The method for preparing the cell culture fluid according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: taking 42.5 mL of DMEM basal culture medium, adding 7.5 mL of serum, adding OTS186935, F5446 and SETDB1-TTD-IN-1, mixing well, and filtering the liquid through a 0.22 μm sterile filter.

7. The method according to claim 6, characterized in that The mass concentration of OTS186935 was 1 μM, the mass concentration of F5446 was 0.25 μM, and the mass concentration of SETDB1-TTD-IN-1 was 1 μM.

8. The method according to claim 6, characterized in that The mass concentration ratio of OTS186935, F5446 and SETDB1-TTD-IN-1 is 4:1:

4.

9. Use of the culture medium according to any one of claims 1 to 3 in improving the in vitro developmental ability of porcine somatic cell nuclear transfer embryos.

10. The use according to claim 9, characterized in that After reducing the H3K9me3 modification of porcine fetal fibroblasts, somatic cell nuclear transfer was performed to improve the in vitro development of somatic cell nuclear transfer.

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