CN108588008A - A kind of black pig handmade cloning in Debao reconstructs drug and the application of vitro Development of Embryos - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a medicine for the in vitro development of Debao black pig manual cloned and reconstituted embryos and its application. It is found that the appropriate concentration (0.25 μmol /L～0.5μmol/L) of 5‑Aza‑CdR can reduce the methylation level of donor nuclear fibroblasts, and increase the blastocyst development rate and the total cell number of blastocysts. ‑Aza‑CdR treated the reconstructed embryos, counted the cleavage rate, blastocyst rate and blastocyst cell number, and found that 20nmol/L, 72h was the best treatment condition. Analysis Since the drug residue and cytotoxicity of 5‑Aza‑CdR itself will have an additive effect on the reconstituted embryos after the donor and the reconstituted embryos are treated successively, an analysis method was determined to achieve the best results.

Description

A kind of medicine and its application for in vitro development of Debao black pig manual cloned and reconstituted embryos

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a medicament for in vitro development of Debao black pig manual cloned and reconstructed embryos and its application.

Background technique

At present, the organic combination of somatic cell nuclear transfer (SCNT) technology and genomics modification technology provides a strong technical support for basic biological research such as the production of transgenic animals, the development of animal models of human genetic diseases, and xenogeneic organ transplantation. Because pigs are very similar to humans in terms of anatomy, tissue, physiology, and nutritional metabolism, they are considered to be the most ideal donors for human xenogeneic nuclear transplantation. Therefore, scientists at home and abroad have focused their attention on the SCNT technology and genome modification technology. combined. Because the cloning efficiency of large livestock, especially pigs, is still very low (1%-2%), it greatly restricts the development of related research work on somatic cell cloning pigs and transgenic cloning pigs. There are many factors that affect the early development of somatic cell cloning reconstituted embryos, such as the coordination of the growth cycle of the donor cell and the recipient cell, the process of nucleoplasm fusion, and whether the nucleoplasm exchange can be successfully completed after the 8-cell stage. , and the DNA methylation and demethylation levels of the reconstituted embryos all affect the normal development of the somatic cell nuclear transfer reconstituted embryos. Epigenetic modification of donor cells and reconstituted embryos can improve the developmental potential of nuclear transfer embryos. DNA methyltransferase inhibitors are epigenetic modifiers widely used in somatic cells. Its methylation and demethylation in SCNT play a decisive role in the reprogramming of donor and recipient genomes. 5-Aza-2'-deoxycytidine (5-Aza-2'-deoxycytidine, 5-Aza-CdR) is an irreversible DNA methyltransferase inhibitor, which can activate methylated silenced genes at low doses, and Cytotoxic dual effects. It has been certified by the US FDA and is one of the commonly used anticancer drugs in clinical practice. At the same time, it is used as a DNA methylase inhibitor. It shows that treating donor cells with 5-Aza-CdR can improve the developmental potential of bovine nuclear transfer embryos in vitro. However, high concentrations of 5-Aza-CdR have also been shown to be cytotoxic to bovine nuclear transfer embryos, while treatment of donor cells with low concentrations of 5-Aza-CdR is beneficial to the development of pig cloned embryos. After the study first treated fibroblast donors with 5-Aza-CdR, it was found that high-dose 5-Aza-CdR can reduce the DNA methylation level of donor cells and reconstituted embryos, but hinder the development of embryos , It was later found that low doses can achieve the purpose of reducing methylation levels and improving blastocyst development. 5-Aza-CdR was used to treat HMC reconstituted embryos of Debao black pigs. Traditional analysis methods could not determine the ratio concentration of 5-Aza-CdR, and could not understand its influence on the in vitro developmental potential of reconstituted embryos.

To sum up, the problems existing in the existing technology are: 5-Aza-CdR treats Debao black pig HMC reconstituted embryos, the traditional analysis method cannot determine the ratio concentration of 5-Aza-CdR; if 5-Aza-CdR Excessive doses can reduce the DNA methylation level of donor cells and reconstituted embryos, but hinder the development of embryos, and the analysis method cannot understand its impact on the in vitro development potential of reconstituted embryos.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a drug and application for in vitro development of Debao black pig manual cloned and reconstructed embryos,

The present invention is achieved in this way, a drug for in vitro development of Debao black pig manual cloned and reconstructed embryos. ;

The concentration of the 5-aza-2′-deoxycytidine is 0.25 μmol/L˜0.5 μmol/L; the treatment is 72 hours.

Further, the concentration of the 5-aza-2'-deoxycytidine was 20nmol/L, and the treatment was performed for 72h.

Another object of the present invention is to provide an application of the medicament for the in vitro development of Debao black pigs manually cloned and reconstituted embryos in the breeding of Debao black pigs.

The present invention treats donor fibroblasts with 5-Aza-CdR for 72 hours and finds that 5-Aza-CdR at an appropriate concentration (0.25 μmol/L-0.5 μmol/L) can reduce the methylation of donor nuclear fibroblasts level, and increased the blastocyst development rate and blastocyst total cell number, treated the reconstructed embryos with different concentrations and different times of 5-Aza-CdR, and counted the cleavage rate, blastocyst rate and blastocyst cell number, and found that 20nmol/L, 72h is the best treatment condition. Analysis Since the drug residue and cytotoxicity of 5-Aza-CdR itself will show superimposed effects on the reconstituted embryo after the donor and the reconstituted embryo are treated successively, the analysis method is determined to achieve the best effect.

Description of drawings

Fig. 1 is a display diagram of HMC blastocysts after treatment of reconstituted embryos with different concentrations of 5-Aza-CdR in the method for analyzing the effect of 5-Aza-CdR on the in vitro development of reconstituted embryos provided by the embodiment of the present invention.

Fig. 2 is a display diagram of Hoechst33342 staining of HMC blastocysts after treatment of reconstituted embryos with different concentrations of 5-Aza-CdR by the method for analyzing the effect of 5-Aza-CdR on the in vitro development of reconstituted embryos provided by the embodiment of the present invention.

Fig. 3 is a display diagram showing Hoechst33342 staining of HMC blastocysts after 5-Aza-CdR treatment of reconstituted embryos for different time in the method for analyzing the effect of 5-Aza-CdR on the in vitro development of reconstituted embryos provided by the embodiment of the present invention.

Fig. 4 is a display diagram of Hoechst33342 staining of HMC blastocysts after simultaneous treatment of donor and reconstituted embryos with different concentrations of 5-Aza-CdR by the method for analyzing the effect of 5-Aza-CdR on the in vitro development of reconstituted embryos provided by the embodiment of the present invention.

Detailed ways

In order to further understand the content, features and effects of the present invention, the following examples are given, and detailed descriptions are given below with reference to the accompanying drawings.

The application principle of the present invention will be further described in conjunction with experiments below.

1 Materials and methods

1.1 Main reagent consumables

Fetal bovine serum (FBS) was purchased from Gbico Company, TCM-199 powder and DMEM powder were purchased from Gbico Company in the United States, hormones were eCG, hCG was purchased from Sigma Company in the United States, and other chemical reagents were purchased from SigmaAldrich Company in the United States unless otherwise specified. Cell Fusion Instrument Model: ECM2001, Fusion Tank Model: BTX 450, Embryo Accumulation Needle Model: DN-10 Type, Oocyte Maturation Petri Dish: 35mm and 60mm Plastic Petri Dish, Self-made Enucleation Knife, Embryo Petri Dish: Four-hole Plate .

1.2 Mature culture of oocytes

The ovaries were obtained from the slaughterhouse in Nanning City, put into normal saline containing double antibodies, and sent back to the laboratory within 4 hours with a thermos. First rinse the ovaries with ordinary saline, then add preheated saline containing double antibodies, and send them back to the embryonic room to extract oocytes. Use a 10mL syringe to extract follicles 2-6mm from the surface of the ovary, put the follicle fluid into a test tube and let it stand, discard the supernatant, and save the precipitated part for subpackaging. Select cumulus-oocyte complexes (COCs) that contain 3 or more layers of granule/cumulus cell-coated and good refraction, and put them into the maturation solution (containing 10IU/mL hCG+10IU/mL eCG) for culture, 20 After ~22h, replace with hormone-free culture medium, and continue culturing for 18~20h until the cells mature.

1.3 Culture of fetal fibroblasts (tissue block culture method)

Fetal pigs were obtained from the slaughterhouse of Nanning City, placed in double-antibody saline, and sent back to the laboratory within 4 hours. Wash it three times with ordinary normal saline, transfer it to a sterile ultra-clean bench, separate part of the thigh tissue, and then wash it twice with PBS containing double antibodies, put it into a 60mm plate, and cut it into 2-3mm tissue Add a small amount of DMEM culture solution containing 10% FBS to the petri dish, spread it evenly with tweezers, mark the dish cover, put it upside down in the incubator, add a small amount of culture solution after 4 hours to prevent tissue Blocks float. After 24 hours, fill up the culture solution at the bottom of the plate, and continue to observe the growth of fibroblasts after 48 hours. When the fibroblasts grow to about 90% confluence, they are passaged and used as donor cells for manual cloning of pigs.

1.4 Oocyte enucleation method

Select high-quality oocytes and place them in the egg washing solution, and blow them several times to remove granulosa cells. Then move it into the pronase solution. When the zona pellucida begins to deform, immediately move it into the washing solution (T20) containing 20% FBS to wash, then put it into T20 until it returns to a round shape, and use a glass pipette to remove the oocyte Transfer to the cutting solution (containing 2.5 μg/mL CB), and select the recovered intact oocytes for enucleation.

Polar body positioning method: pick the cells with complete first polar body, place the polar body at the position of 6 o’clock or 12 o’clock, cut off about 1/3 of the egg cytoplasm in the direction of the polar body with a cutting knife, and keep the enucleated cytoplasm for future use follow-up test.

1.5 Fusion/activation of donor/recipient cells

A one-step fusion method is used. Fusion/activation is a three-step process, first bonding, then fusion/electrical activation, and finally chemical activation.

Adhesion: First use phytohemagglutinin (PHA) to bond the non-nucleated half-egg and the donor cell to form a half-egg-donor cell pair; Egg-donor-egg-like adhering bodies are transferred into the electrofusion solution to be fused.

Fusion/Electroactivation: Put the bonded body evenly into the fusion tank with electrofusion solution. The electrofusion parameters are: AC: 6V/cm; DC: 1.2kV/cm, 30μs, 2DC. After the electrical activation is completed, put it into the incubator to recover for 30 minutes, and then check its fusion.

Chemical activation: put the fused reconstituted embryos into PZM-3 activation solution containing 5 μg/mL CB and 10 μg/mL CHX for activation, put one reconstituted embryo into each drop of activation solution, and activate for 4 hours.

1.6 Microcavity culture of reconstituted embryos

In order to ensure that HMC reconstituted embryos without zona pellucida can grow in a relatively independent environment, this study adopts the micro-well culture method. The activated embryos were transferred to microwells in PZM-3 that had been equilibrated in a CO ₂ incubator for at least 4 h and covered with paraffin oil. Preparation of micro-cavities: Use an embryo gathering needle to uniformly rotate micro-cavities at the bottom of the four-hole plate. The size of the micro-cavities is set according to the requirements of the experiment, and one embryo is placed in each micro-cavity. Observe the cleavage rate at 48h, and observe the blastocyst rate at 160-168h.

1.7 Blastocyst cell count

Collect the blastocysts on the 6th to 7th day, wash them in CCM for 2 to 3 times, place them in 10 μmol/mL Hoechst33342 dye solution, and stain them in the dark at room temperature for 15 minutes, take them out and wash them with CCM for 2 to 3 times, paraffin- Cover the slides with petroleum jelly, observe and record the total number of blastocyst cells under a fluorescence microscope.

1.8 Experimental design

Experiment 1: Comparing the effects of 40nmol/L, 20nmol/L, 10nmol/L, 5nmol/L, 0nmol/L four concentrations of 5-Aza-CdR treatment of HMC reconstituted embryos for 72h on their developmental effects;

Experiment 2: Comparing the effects of 20nmol/L 5-Aza-CdR treatment on HMC reconstituted embryos for 96h, 72h, 48h, 24h, and 0h on the developmental effect;

Experiment 3: Comparing the four treatment groups of 1μmol/L, 0.5μmol/L, 0.25μmol/L, 0μmol/L and the optimal concentration of 20nmol/L for the reconstituted embryos to treat the donor and the reconstituted embryos at the same time, the effect on their development influences;

1.9 Statistics

The obtained data were statistically analyzed by SPSS 17.0 software. The same batch of experiments was performed every time the experiment was repeated, and the experiments were carried out under the same source of ovaries and experimental conditions. All experiments were repeated at least 3 times.

2 result analysis

2.1 Effects of different concentrations of 5-Aza-CdR on the in vitro development of HMC embryos treated with reconstituted embryos

Comparing the effects of 40nmol/L, 20nmol/L, 10nmol/L, 5nmol/L, 0nmol/L four concentrations of 5-Aza-CdR on HMC reconstituted embryos for 72h, the results showed that: 5-Aza-CdR Treatment for 72 hours can increase the division rate, morula rate, blastocyst rate and blastocyst cell number of reconstituted embryos. For each treatment group, the 20nmol/L treatment group can significantly increase the blastocyst rate, 10nmol/L and 20nmol/L The L treatment group could significantly increase the number of blastocyst cells, and there was no significant difference between the two groups. See Table 1 for details. See Figure 1 for the development of blastocysts on the seventh day, and Figure 2 for Hoechst33342-stained blastocyst cells.

Table 1 Effects of different concentrations of 5-Aza-CdR on the in vitro development of HMC embryos treated with reconstituted embryos

Different superscript letters in the same column indicate significant difference (P<0.05).

2.2 Effects of 5-Aza-CdR treatment of reconstituted embryos at different times on the in vitro development of HMC embryos

20nmol/L 5-Aza-CdR was used to treat HMC reconstituted embryos at different times (96h, 72h, 48h, 24h, 0h), and the embryonic development at each stage was counted. It was found that 5-Aza-CdR treatment could effectively improve the reconstituted embryo. Splitting rate, morula rate, blastocyst rate and blastocyst cell number, wherein the 72-hour treatment group can significantly improve blastocyst rate and blastocyst cell number, see Table 2 and Figure 3 for details.

Table 2 Effects of 220nmol/L 5-Aza-CdR treatment of reconstituted embryos at different times on the in vitro development of HMC embryos

Different superscript letters in the same column indicate significant difference (P<0.05).

2.3 Effects of 5-Aza-CdR treatment on donor and reconstituted embryos on the in vitro development of HMC embryos

The donors and the reconstituted embryos were simultaneously treated with four treatment groups of 0 μmol/L, 0.25 μmol/L, 0.5 μmol/L and 1 μmol/L and the optimal concentration of 20 nmol/L of the reconstituted embryos, the results are shown in Table 3, each treatment Compared with the control group, the developmental rate of reconstructed embryos and the total number of blastocysts in the group were not significantly increased, but even decreased. The analysis may be due to the adverse effects on the later development of embryos caused by improper treatment concentration and time when the donor and reconstructed embryos were treated at the same time. , see Table 3 and Figure 4 for details.

Table 3 Effects of simultaneous treatment of donor and reconstituted embryos with different concentrations of 5-Aza-CdR on the in vitro development of MHC embryos

Treatment group (donor treatment + reconstituted embryo treatment): 1:0μmol/L+20nmol/L; 2:0.25μmol/L+20nmol/L; 3:0.5μmol/L+20nmol/L; 4:1μmol/L +20nmol/L.

Different superscript letters in the same column indicate significant difference (P<0.05).

The present invention treats donor fibroblasts with 5-Aza-CdR for 72 hours and finds that 5-Aza-CdR at an appropriate concentration (0.25 μmol/L-0.5 μmol/L) can reduce the methylation of donor nuclear fibroblasts level, and increased the blastocyst development rate and the total number of blastocyst cells. It was found that 20nmol/L, 72h was the best treatment condition. Finally, after treating with the best treatment conditions of the donor and embryo explored earlier, it was found that The treatment of 5-Aza-CdR did not promote the development of embryos. The analysis may be due to the fact that the drug residue and cytotoxicity of 5-Aza-CdR itself will show superimposed effects on the reconstructed embryos after successive treatments of the donor and the reconstructed embryos. effect. Therefore, if the purpose of reducing DNA methylation level and improving embryonic development is to be achieved during simultaneous treatment, it is necessary to adjust the respective treatment concentration and time in future experiments in order to achieve the best effect. Treatment of donor cells with 5-Aza-CdR at an appropriate concentration (0.25 μmol/L-0.5 μmol/L) for 72 hours and treatment of reconstituted embryos with 20 nmol/L 5-Aza-CdR for 72 hours can reduce the level of DNA methylation and effectively Improving the developmental potential of HMC embryos in Debao black pigs.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Any simple modifications made to the above embodiments according to the technical essence of the present invention, equivalent changes and modifications, all belong to this invention. within the scope of the technical solution of the invention.

Claims (3)

1. a kind of drug of the black pig handmade cloning reconstruct vitro Development of Embryos in Debao, which is characterized in that the black pig in Debao is manual The drug of clone's reconstruct vitro Development of Embryos is 5-aza-2'-deoxycytidine；

0.25 μm of ol/L~0.5 μm ol/L of the 5-aza-2'-deoxycytidine concentration；Handle 72h.

2. the drug of the black pig handmade cloning reconstruct vitro Development of Embryos in Debao as described in claim 1, which is characterized in that described A concentration of 20nmol/L of 5-aza-2'-deoxycytidine handles 72h.

3. a kind of drug of the black pig handmade cloning reconstruct vitro Development of Embryos in Debao as described in claim 1 is in the black pig cultivation in Debao In application.