Method for improving development efficiency of pig cloned embryo by reducing H3K9me3 modification of pig cloned embryo and application

Abstract

The invention discloses a method for improving the development efficiency of a pig cloned embryo by reducing H3K9me3 modification of the pig cloned embryo and application, and belongs to the technical field of embryo culture. The invention aims to improve the in-vitro development efficiency of the pig cloned embryo. The invention provides a culture solution for improving the in-vitro developmental capacity of a pig cloned embryo. The culture solution is prepared from the following components: a PZM-3 culture medium serving as a basic culture solution, OTS186935 and F5446. The scheme provided by the invention has the large-scale application advantages of low cost, stability improvement and the like, and an innovative solution is provided for large-scale cloning of excellent boars.

Description

Technical Field

[0001] The present invention belongs to the technical field of embryo culture, and particularly relates to a method and application for improving the development efficiency of pig cloned embryos by reducing H3K9me3 modification. Background Art

[0002] Somatic cell nuclear transfer (SCNT) involves using micromanipulation techniques to inject a single nucleus into the cytoplasm of an enucleated oocyte, creating a cloned embryo. Once inside the oocyte, the somatic cell nucleus is reprogrammed by factors in the oocyte's cytoplasm to become the nucleus of a fertilized egg. The production of cloned pigs using SCNT holds great promise for applications in animal husbandry and biomedicine. However, low cloning efficiency has been a key bottleneck hindering its widespread application.

[0003] To date, the development of porcine embryo cloning technology still faces challenges such as low cloning efficiency, low survival rates of cloned embryos cultured in vitro, and high rates of malformations, which to some extent limit its advancement. The developmental efficiency of cloned embryos is far lower than that of embryos fertilized in vitro. The efficiency of early development of cloned mammalian embryos is also low, with the cloned blastocyst rate reaching only 10-15%, compared to fertilized blastocyst rates of 30-80%. Improper micromanipulation techniques and an imperfect in vitro culture system for cloned embryos are also major factors contributing to low cloning efficiency. Furthermore, errors in epigenetic reprogramming of the donor cell nucleus can also lead to low cloning efficiency and abnormal development of cloned embryos.

[0004] Notably, cloned embryos still exhibit developmental impairments during maternal to zygotic genome activation (ZGA), which is thought to result from incomplete epigenetic reprogramming in the somatic cell genome.

[0005] Injection of porcine KDM4A mRNA can effectively reduce this modification and significantly increase the blastocyst rate of cloned embryos. The economic cost of siRNA and mRNA synthesis is high, they are unstable, easily degraded, and have a dosage effect. On the other hand, injection has high technical requirements for personnel and low efficiency, and the injection operation will cause damage to the embryo. Summary of the Invention

[0006] The purpose of the present invention is to improve the efficiency of in vitro development of pig cloned embryos.

[0007] The invention provides a culture solution for improving the in vitro development ability of pig cloned embryos. The culture solution consists of the following components: PZM-3 culture medium as a basic culture solution, OTS186935 and F5446.

[0008] The invention provides a culture solution for improving the in vitro development ability of pig cloned embryos. The culture solution comprises the following components: DMEM culture medium as a base culture solution, OTS186935 with a mass concentration of 0.05-0.2 μM and F5446 with a mass concentration of 0.05-0.2 μM.

[0009] The invention provides a culture solution for improving the in vitro development ability of pig cloned embryos. The culture solution comprises the following components: DMEM culture medium as a base culture solution, OTS186935 with a mass concentration of 0.2 μM and F5446 with a mass concentration of 0.2 μM.

[0010] The invention provides a culture solution for improving the in vitro development ability of pig cloned embryos. The culture solution comprises the following components: DMEM culture medium as a base culture solution, OTS186935 with a mass concentration of 0.05 μM and F5446 with a mass concentration of 0.05 μM.

[0011] The invention provides a culture solution for improving the in vitro development ability of pig cloned embryos. The culture solution consists of the following components: DMEM culture medium is used as the basic culture solution, and the mass concentration of OTS186935 and F5446 is 1:1.

[0012] The present invention provides a method for preparing the above-mentioned culture solution, and the steps of the method are as follows: taking 9.7 mL of PZM3 mother liquid, preparing OTS186935 and F5446, mixing, and filtering the liquid with a 0.22 μm sterile filter.

[0013] It is further defined that the mass concentration of OTS186935 and F5446 is 1:1.

[0014] The present invention provides an application of the above-mentioned culture solution in improving the in vitro development ability of pig cloned embryos.

[0015] The present invention provides a method for improving the in vitro development ability of pig cloned embryos, wherein the pig somatic cell nuclear transplanted embryos are cultured in the above-mentioned culture medium for 30-84 hours.

[0016] Further defined, the culture conditions were 5% CO2 and 95% air at 39°C.

[0017] Beneficial effects: The present invention provides a porcine cloned embryo culture medium based on epigenetic regulation and its application, which belongs to the field of animal embryo engineering technology. The culture medium forms a composite inhibitor system by adding specific histone methyltransferase inhibitors OTS186935 (0.1-10uM) and F5446 (0.01-5uM) to the PZM-3 culture medium. The two small molecule inhibitors OTS186935 and F5446 in the present invention can synergistically reduce the abnormal modification level of H3K9me3 in cloned embryos, effectively promote the transcriptional activation of porcine cloned embryos, and significantly improve the in vitro development efficiency of somatic cell nuclear transplanted embryos. The blastocyst rate of porcine cloned embryos can be relatively increased by 72% (from 14.4% to 24.8%), and the blastocyst diameter can be increased by 57.8% (from 60.76μm to 95.93μm). Compared with the existing mRNA injection method, this solution has the advantages of large-scale application such as simple operation (direct addition of culture medium), low cost, and improved stability, providing an innovative solution for the large-scale cloning of excellent breeding pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 H3K9me3 histone modification in parthenogenetic embryos at the 4C stage;

[0019] Figure 2 H3K9me3 histone modification in parthenogenetic embryos at the 8C stage;

[0020] Figure 3 H3K9me3 histone modification in cloned embryos at the 4C stage;

[0021] Figure 4 H3K9me3 histone modification in cloned embryos at the 8C stage;

[0022] Figure 5 EU staining of cloned embryos at 4C;

[0023] Figure 6 Analysis of transcriptional status in porcine cloned embryos treated with the inhibitors OTS186935 and F5446 at the 4-8C stage; A is the principal component analysis, B is the heat map analysis result; C is the expression level analysis result;

[0024] Figure 7 .In vitro development of cloned embryos cultured with PZM-3 supplemented with two inhibitors, OTS186935 and F5446; A is an electron micrograph of blastocyst development; B is a statistical graph showing the blastocyst rate, C is a statistical graph showing the blastocyst diameter, and D is a statistical graph showing the blastocyst cell number. DETAILED DESCRIPTION

[0025] 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.

[0026] Reagent sources: All experimental reagents in this invention were purchased from Sigma (USA), except for those specified: rabbit anti-H3K9me3 antibody (AB8898), secondary antibodies (Invitrogen, A21207 and A11008), BeyoClick TM EU-488 RNA Synthesis Detection Kit (Biyuntian R0301S).

[0027] OTS186935 (MCE, HY-122181) and F5446 (MCE, HY-150190).

[0028] Basic culture medium is required for in vitro maturation of oocytes, retrieval of mature oocytes, and preparation for somatic cell nuclear transfer. The formula of the culture medium is as follows:

[0029] Table 1 Hepes culture medium

[0030]

[0031] Table 2. Oocyte maturation fluid mother fluid

[0032]

[0033]

[0034] Table 3 Mature oocyte operating fluid

[0035]

[0036] Table 4FM fusion solution

[0037]

[0038] Table 5 PZM-3 mother liquor

[0039]

[0040] Example 1. A culture medium for improving the in vitro developmental ability of pig cloned embryos

[0041] 1. Use PZM-3 medium as the base culture medium, with a mass concentration of 0.05 μM OTS186935 and a mass concentration of 0.05 μM F5446.

[0042] Take 9.7 mL of PZM3 stock solution and prepare 0.05 μM OTS186935 + 0.05 μM MF5446-PZM3 culture system according to the additives in the table below. After thorough mixing, filter the liquid through a 0.22 μm sterile filter.

[0043] Table 6 0.05 μm OTS186935 + 0.05 μm F5446-PZM3 culture system components

[0044]

[0045] Example 2. A culture medium for improving the in vitro developmental ability of pig cloned embryos

[0046] 1. Use PZM-3 medium as the base culture medium, with a mass concentration of 0.2μM OTS186935 and a mass concentration of 0.2μM F5446.

[0047] 2. Take 9.7 mL of PZM3 stock solution and prepare 0.2 μM OTS186935 + 0.2 μM MF5446-PZM3 culture system according to the additives in the table below. After thorough mixing, filter the liquid through a 0.22 μm sterile filter.

[0048] Table 7 0.2 μm OTS186935 + 0.2 μm F5446-PZM3 culture system components

[0049]

[0050] Table 8 In vitro development statistics of SCNT embryos cultured with PZM-3 supplemented with two inhibitors, OTS186935 and F5446

[0051]

[0052] Example 3.

[0053] 1. Obtaining pig cloned embryos

[0054] 1. In vitro oocyte maturation: Porcine ovaries collected from the slaughterhouse were transported to the laboratory within 1 hour of being placed in 37°C saline. After the ovaries were temperature-tested, they were gently drained with a strainer and then rinsed with 37°C saline supplemented with double-antibody antibodies. Using a 10mL syringe with a 10G needle, COCs were extracted from follicles 2 to 8 mm in diameter. The extracted follicular fluid was placed in a 50mL conical-bottom centrifuge tube in a 37°C water bath. After oocyte retrieval, the centrifuge 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 solution. The tube was shaken and allowed to settle for 3 minutes in an incubator. The supernatant was discarded. This wash was repeated twice. Hepes solution was added for the third time, and the tube was transferred to a large domestic petri dish for oocyte collection. Under a stereomicroscope, COCs containing intact cumulus cells and homogeneous cytoplasm were selected and cultured in maturation medium at 38.5°C, 5% CO2, 95% air, and saturated humidity for 42–44 hours.

[0055] 2. Obtaining mature oocytes: After culturing the COCs in step 1 for 42-44 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, select mature oocytes based on the release of the first polar body as the criterion for oocyte maturity.

[0056] 3. Preparation and in vitro culture of porcine cloned embryos: From the mature oocytes in step 2, oocytes with distinct polar bodies and uniform cytoplasm were selected as recipients. The oocyte nucleus was removed by aspirating the first polar body and adjacent cytoplasm through a glass tube. A single porcine embryonic fibroblast (PEF) was then injected into the perivitelline space of the oocyte using an injection needle. The reconstructed embryos were then fused by electric shock using two direct pulses of 120 V / mm for 30 ms in the fusion medium. The reconstructed embryos were then cultured in PZM-3 medium at 39°C in an atmosphere of 5% CO2 and 95% air.

[0057] 2. Effects of Adding OTS186935 and F5446 to the Porcine Nuclear Transfer Embryo Culture Medium

[0058] The porcine cloned embryos from Example 2 were cultured for 30 h and then transferred to PZM-3 culture medium containing 0.1 μM OTS186935 and 0.1 μM F5446. After culture for 84 h, they were transferred to PZM-3 culture medium again. During the culture period, they were placed in an incubator at 38.5°C, 5% CO2, and saturated humidity. Blastocysts were obtained on day 6.5.

[0059] Comparative Example 1:

[0060] The porcine cloned embryos described in Example 3 were cultured in PZM-3 culture medium in an incubator maintained at 38.5°C, 5% CO2, and saturated humidity. ZGA (zygotic genome activation) samples were obtained after 84 hours of culture, and blastocyst samples (control group) were obtained after 6.5 days of culture.

[0061] Effect experiment:

[0062] (1) Immunofluorescence detection

[0063] The cell samples and ZGA stage or blastocyst samples obtained in Example 2 and Comparative Example 1 were placed in a 96-well plate, fixed with 4% paraformaldehyde solution, permeabilized with 1% Triton X-100 in PBS, and fixed with solution (1% BSA in PBS) for 1 hour. Immunolabeling The embryos were incubated at 4°C overnight, washed three times with an anti-H3K9me3 primary antibody, and incubated with a secondary antibody for 1 hour, diluted 1:2000 with fixative solution. The samples were washed and stained with 5 mg / mL Hoechst 33342. Fluorescence was detected and imaged using a Nikon fluorescence microscope. The average optical intensity (AOI) of H3K9me3 staining was calculated using ImageJ software. H3K9me3 staining in the nuclear area was subtracted by AOI staining. Finally, the statistical analysis data were analyzed using SPSS software.

[0064] (2) 5-EU staining

[0065] Take the 4-cell embryo and press BeyoClick TM EU-488 kit procedures: 2× EU working solution preheated at 37°C was mixed with an equal volume of culture medium, and the embryos were immersed and incubated for 30 minutes (37°C, 5% CO2). The embryos were fixed with 4% paraformaldehyde at room temperature for 30 minutes and washed three times with PBS-T. The embryos were permeabilized with 0.5% Triton X-100 for 15 minutes and labeled with Click reaction solution (containing Alexa Fluor 488) in the dark for 45 minutes. Nuclear counterstaining was performed with 5 μg / mL Hoechst 33342 for 10 minutes. After antifade removal and mounting, the embryos were imaged using a Nikon fluorescence microscope (Hoechst: Ex 405 / Em 450 nm; EU-488: Ex 488 / Em 525 nm). The mean fluorescence intensity of EU in the cytoplasmic region (nuclear signal subtracted) was analyzed using ImageJ, and statistical analysis was performed using SPSS (ANOVA, p < 0.05).

[0066] (3) In vitro development efficiency of pig cloned embryos

[0067] The blastocyst rates (number of blastocysts / total number of nuclear transplants×100%) after 6.5 days of culture in Example 2 and Comparative Example 1 were counted, and the blastocyst diameters and blastocyst cell numbers were counted.

[0068] result:

[0069] 1) Effect of OTS186935 and F5446 inhibitors added to PZM-3 culture medium on H3K9me3 modification in parthenogenetic embryos at the ZGA stage in vitro. 30-84 hours after activation, 0.1uM OTS186935 and 0.1uM F5446 inhibitors were added to the parthenogenetic embryos and cultured. Samples were harvested at 4-8°C for IF staining of H3K9me3.

[0070] The results showed that the H3K9me3 histone modification level of PA-4C and PA-8C embryos obtained from the experimental group was significantly reduced ( Figure 1 and Figure 2 ).

[0071] 2) Effect of OTS186935 and F5446 inhibitors on H3K9me3 modification in ZGA stage somatic cell nuclear transfer embryos in PZM-3 culture medium. 30-84h after SCNT activation, 0.1uM OTS186935 and 0.1uM F5446 inhibitors were added to culture nuclear transfer embryos, and samples were collected 4-8C for IF staining of H3K9me3.

[0072] The results showed that the H3K9me3 histone modification level of SCNT-4C and SCNT-8C embryos obtained from the experimental group was significantly reduced ( Figure 3 and Figure 4 ).

[0073] 3) 5-EU staining results: The experimental results showed that the green fluorescence intensity of the experimental group (EU-OF) (88.32±0.65) was significantly higher than that of the control group (EU-con) (30.25±1.59, P=0.007) ( Figure 5 ).

[0074] This result showed that the transcription level of OF-4C embryos in the experimental group was significantly higher than that in the 4C embryos in the control group, suggesting that the combined treatment of OTS186935 and F5446 can effectively promote the transcriptional activation of pig cloned embryos ( Figure 6 ).

[0075] 4) Effects of adding OTS186935 and F5446 inhibitors to PZM-3 culture medium on the development of somatic cell nuclear transfer embryos

[0076] 0.1uM OTS186935 and 0.1uM F5446 inhibitors were added to the nuclear transplanted embryos 30-84h after SCNT activation, and the blastocyst rate and blastocyst diameter were counted after culture until D6.5.

[0077] The results showed that there was no significant difference in the cleavage rate between the experimental group and the control group, but the blastocyst rate in the experimental group increased by 10.37% (Table 9, Figure 7 In addition, the diameter of the blastocyst increased significantly (Table 9 Figure 7 C, D in ).

[0078] Table 9. In vitro development statistics of SCNT embryos cultured with PZM-3 supplemented with OTS186935 and F5446 inhibitors

[0079]

Claims

1. A culture medium for improving the in vitro developmental ability of porcine cloned embryos, characterized in that: The culture solution consists of the following components: PZM-3 culture medium as the base culture solution, OTS186935 and F5446.

2. A culture medium for improving the in vitro developmental ability of porcine cloned embryos, characterized in that: The culture solution consists of the following components: DMEM culture medium as the base culture solution, OTS186935 with a mass concentration of 0.05-0.2 μM and F5446 with a mass concentration of 0.05-0.2 μM.

3. A culture medium for improving the in vitro developmental ability of porcine cloned embryos, characterized in that: The culture solution consists of the following components: DMEM culture medium as the base culture solution, OTS186935 with a mass concentration of 0.2 μM and F5446 with a mass concentration of 0.2 μM.

4. A culture medium for improving the in vitro developmental ability of porcine cloned embryos, characterized in that: The culture solution consists of the following components: DMEM culture medium as the base culture solution, OTS186935 with a mass concentration of 0.05 μM and F5446 with a mass concentration of 0.05 μM.

5. A culture medium for improving the in vitro developmental ability of porcine cloned embryos, characterized in that: The culture solution consists of the following components: DMEM culture medium is used as the base culture solution, and the mass concentration of OTS186935 and F5446 is 1:

1.

6. The method for preparing the culture solution according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: taking 9.7 mL of PZM3 mother liquor, preparing OTS186935 and F5446, mixing them evenly, 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 and F5446 is 1:

1.

8. Use of the culture medium according to any one of claims 1 to 5 in improving the in vitro developmental ability of porcine cloned embryos.

9. A method for improving the in vitro developmental ability of porcine cloned embryos, characterized in that: The porcine somatic cell nuclear transplanted embryo is cultured in the culture medium according to any one of claims 1 to 5 for 30 to 84 hours.

10. The method according to claim 9, characterized in that The culture conditions were 5% CO2 and 95% air at 39°C.

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