A culture medium for promoting in vitro development of porcine somatic cell cloned embryos

A technology for cloning embryos and in vitro development, applied to artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of incompleteness, slow embryonic state, low cloning efficiency, etc., and achieve short half-life and high biological safety , the effect of increasing the quantity

Active Publication Date: 2016-12-07
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, the oocyte itself reprograms sperm chromosomes rather than somatic cells, so the reprogramming effect guided by nuclear transfer is not very ideal, and the reprogramming of highly differentiated cells to the embryonic state is slow and relatively incomplete (X.C.Tian et al., Nuclear reprogramming by somatic cell nuclear transfer-the cattle story, Society of Reproduction and Fertility supplement, 64(2007):327-339), which may be due to incomplete reprogramming of somatic cells and apparent Due to abnormal changes in genetics, the efficiency of animal somatic cell cloning is still low

Method used

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  • A culture medium for promoting in vitro development of porcine somatic cell cloned embryos
  • A culture medium for promoting in vitro development of porcine somatic cell cloned embryos

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1. Preparation of porcine oocyte maturation culture medium

[0024] Add 10% porcine follicular fluid, 5% fetal bovine serum, 10IU / mL pregnant horse serum gonadotropin, 10IU / mL human chorionic gonadotropin, 10ng / mL epidermal growth factor, Prepared with 100IU / mL penicillin and 100μg / mL streptomycin.

Embodiment 2

[0025] Example 2. Acquisition and in vitro maturation of pig oocytes

[0026] Put the pig ovary just removed from the slaughterhouse into 37°C normal saline containing penicillin and streptomycin, and transport it back to the laboratory within 2 hours; the retrieved ovary is sprayed with 75% alcohol for disinfection once, and sterile normal saline Wash 3 times, and then use a 10mL disposable plastic syringe equipped with a 18-gauge needle to extract follicles with a diameter of 2 to 6mm on the ovary, and inject the extracted liquid into a 15mL centrifuge tube placed in a constant temperature water bath at 38.5°C. Place the centrifuge tube on a constant temperature heating platform at 38.5°C for 15 minutes to allow the cumulus-oocyte complexes (COCs) to settle naturally; discard the supernatant in the centrifuge tube and add egg washing solution (DPBS buffer containing 0.01% PVA) to dilute and mix the precipitate, and quickly pick up COCs with uniform cytoplasm, more than two l...

Embodiment 3

[0028] Example 3. Obtaining porcine MII stage oocytes and preparing donor cells for nuclear transfer

[0029] Transfer the mature COCs after 42±2 hours into the DPBS buffer containing 1 mg / mL Hya hyaluronidase that has been preheated at 38.5°C, and then gently and repeatedly pipette 200 times or about 3 minutes with a pipette. Afterwards, check under the microscope whether the cumulus cells around the COCs have been completely removed, transfer the oocytes that have been removed from the cumulus into T2 operating solution (TCM199 containing 2% fetal bovine serum), wash 3 times, and transfer to mineral oil to cover In the T2 drops; select oocytes with obvious perivitelline space, complete egg cell membrane, and discharge of the first polar body, which are MII stage oocytes.

[0030] The nucleus donor cells are pig fetal fibroblasts or ear fibroblast somatic cells that have been cultured to the 3rd to 9th passages, and the cells with a confluence of 70% to 90% are selected. One ...

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Abstract

The invention provides a culture solution for promoting the in-vitro growth of porcine somatic cell cloned embryos. The culture solution contains an epigenetic modification agent EPZ00477. The EPZ00477 can specifically change the methylation modification level of histone H3K79 in the early stage growth process of cloned embryos to improve the deficiency of oocytes for somatic cell nuclear reprogramming. The culture solution can be used for carrying out in-vitro culture on the porcine somatic cell cloned embryos in order to promote the growth rate and the growth quality of blastulas of the embryos, so the production cost of somatic cell cloned pigs is reduced, the production efficiency of the somatic cell cloned pigs is improved, and it is helpful for further developing and applying a cloning technology.

Description

technical field [0001] The invention relates to an animal embryo culture solution, in particular to a culture solution for promoting the in vitro development of pig somatic cell cloned embryos. Background technique [0002] Somatic cell cloning of animals refers to the use of certain equipment and technical means to recombine animal somatic cells and oocytes from which the nuclear genetic material has been removed, that is, somatic cell nuclear transfer, and then culture the cloned embryos in vitro to a specific developmental stage. After the period, it is then transplanted into the uterus of a surrogate mother in the same physiological state to complete the process of development and production of offspring that are genetically homogeneous with the somatic cell donor nucleus. For more than ten years, somatic cell cloning technology has combined molecular biology and cell culture technology to produce a large number of genetically modified offspring, including animals with r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 张运海张宇陶佳吴蓉花刘星李运生章孝荣
Owner ANHUI AGRICULTURAL UNIVERSITY
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