A kind of preparation method of pig cloned embryo

A technology for cloning embryos and fetuses, which is applied in the field of preparation of pig cloned embryos, can solve the problems of complicated operation process, long working cycle, unfavorable promotion and application of cloned pigs, etc., and achieves improvement of development efficiency, blastocyst rate and favorable promotion. and applied effects

Inactive Publication Date: 2015-12-30
SUN YAT SEN UNIV
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  • Description
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Problems solved by technology

At present, studies suggest that when using reprogramming factors to improve the development efficiency of cloned embryos, it is necessary to integrate the reprogramming factors into the genome of donor cells to effectively increase the level of reprogramming factors in cloned embryos, which requires transfection of donor cells containing After the reprogramming factor carrier, the donor cells need to be further cultured and screened to obtain the nuclear donor cells integrated with the reprogramming factor gene, resulting in a complex operation process and a long working cycle, which is not conducive to the promotion and application of cloned pigs

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  • A kind of preparation method of pig cloned embryo
  • A kind of preparation method of pig cloned embryo
  • A kind of preparation method of pig cloned embryo

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preparation example Construction

[0049] The invention discloses a method for preparing pig cloned embryos. Those skilled in the art can refer to the content of this article, implement the preparation method, and obtain pig cloned embryos. It should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. Inside. The preparation method of the present invention has been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the preparation method herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention .

[0050] All the reagents and raw materials used in the cloned pig embryo provided by the invention can be purchased from the market.

[0051] Configuration of materials and reagents:

[0052] Cell culture medium: DMEM high gluco...

Embodiment 1

[0074] Example 1 Preparation of nuclear donor cells

[0075] In this embodiment, pig fetal fibroblasts are used as donor cells, and the preparation method of the donor cells is as follows:

[0076] Establishment of Porcine Fetal Fibroblast Cell Line (PEF Cell)

[0077]Take out the fetus from the uterus of a gestational sow for 32 days, wash it with 10 mL of DPBS buffer containing double antibodies three times; use sterilized ophthalmic scissors to remove the head, limbs and viscera of the fetus in an ultra-clean workbench, and rinse with DPBS buffer Then transfer to a new petri dish; use another pair of sterilized ophthalmic scissors to cut up the remaining part, and transfer the tissue fragments to a 50mL centrifuge tube. Add 5mL of 0.25% trypsin to the above centrifuge tube, blow and mix with a 1mL gun, and digest at 37°C for 4h. After digestion, transfer the liquid part to a 15mL centrifuge tube, centrifuge at 2000rpm for 15 minutes; discard the supernatant, add 5mLDPBS t...

Embodiment 2

[0084] The preparation of embodiment 2 pig cloned embryos

[0085] Oocyte collection and in vitro maturation culture

[0086] Collect pig ovaries from the slaughterhouse, put them in a thermos bottle filled with 0.9% normal saline (the temperature of the thermos bottle is maintained at 30-35°C), and transport them back to the laboratory within 2 hours; wash the ovaries with 37°C normal saline for 3-4 Use a No. 11 scalpel blade to cut open the follicles on the surface of the ovary to let the follicle fluid flow out, and wash the ovary with TL-HEPES-PVA egg washing solution to collect the follicle fluid and washing fluid. Take 4 mL of follicular fluid and select oocyte-cumulus cell complexes (COCs) with uniform cytoplasm and multi-layered cumulus cells (at least three layers) under a stereomicroscope; wash the obtained COCs twice with TL-HEPES-PVA, Then wash three times with basic medium; then transfer to 0-22 hour medium, culture at 38.5°C, 5% CO2 for 22 hours; transfer to 22-...

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Abstract

The invention belongs to the technical field of biological engineering and discloses a preparation method of pig cloning embryos. The preparation method comprises the following steps of: obtaining donor cells; taking recombinant vectors of reprogramming factors, transferring into the donor cells and obtaining nuclear donor cells; transplanting the nuclear donor cells into egg cells with removed cell nucleuses, and carrying out fusion and activation to obtain the pig cloning embryos. The preparation method provided by the invention has the advantages that the nuclear donor cells are directly transplanted into egg cells with removed cell nucleuses after being obtained, the step of utilizing the reprogramming factors to prepare the pig cloning embryos is reduced, the operation is simple and easy, the working period is shortened and the growing efficiency of the pig cloning embryos is effectively improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for preparing pig cloned embryos. Background technique [0002] Cloned pigs have important application value in animal husbandry production, basic science and medical research. For example, in animal husbandry production, they can preserve and enrich pig breeds; in medical research, pigs can be used for human xenogeneic organ transplantation, disease models and new drugs. Screening provides ideal material. Currently, somatic cell cloning is widely used in the preparation of cloned pigs. [0003] Somatic cell cloning technology refers to the method of nuclear transfer, using cell disassembly or cell recombination technology to enucleate oocytes as nuclear receptors, and use somatic cells or nuclei with a small amount of cytoplasm, namely nuclear plastids, as nuclear donors. Transferring a nuclear donor into a nuclear recipient to construct a recombinant embryo, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/877C12N5/10
Inventor 冀倩倩赵海静宋振威赵广银丛佩清陈瑶生
Owner SUN YAT SEN UNIV
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