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Method for improving efficiency of porcine nuclear transfer

A technology of nuclear transfer and somatic cell nuclear transfer, which is applied in the field of improving the efficiency of pig nuclear transfer, can solve the problems of low expression level, low development efficiency of cloned embryos, low acetylation, etc., and achieves the advantages of simple method, improved in vitro development efficiency and obvious effect Effect

Active Publication Date: 2018-11-30
WENS FOOD GRP CO LTD
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Problems solved by technology

The study found that during the development of cloned embryos, the key genes related to embryonic development were not activated or the expression levels were too low, and the embryos showed excessive methylation and low acetylation levels, indicating that genome reprogramming was incomplete, which was the main reason for the low efficiency of cloned embryo development. reason

Method used

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  • Method for improving efficiency of porcine nuclear transfer
  • Method for improving efficiency of porcine nuclear transfer
  • Method for improving efficiency of porcine nuclear transfer

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example One (+)-JQ1 Different Concentrations to Improve Nuclear Transfer Efficiency

[0023] Take porcine nuclear transfer embryo construction as an example.

[0024] 1. Isolation and culture of donor cells

[0025] The donor cells are from Guangdong Wenshi Group's premium boar ear tissue. After cutting the pig ear skin, put it in DMEM culture solution and store it at 4℃ and transport it back to the laboratory. After the pig ear skin tissue is cut into pieces, the tissue fragments are washed with DMEM Resuspend with appropriate amount of fetal bovine serum and transfer to a petri dish, 37℃, 5% CO 2 , Cultivate in saturated humidity environment. After 5-7h, add DMEM culture medium containing 10% fetal bovine serum, and pass the culture when the cells grow to 90% confluence. On the second day of passaging, change to cell culture medium (DMEM medium + 10% fetal bovine serum) containing different concentrations of (+)-JQ1, treat for 72 hours, and change the medium every 24 hours...

Embodiment 2

[0036] Example 2 The influence of culture medium containing (+)-JQ1 on the efficiency of nuclear transplantation for different periods of donor cell culture

[0037] This embodiment still takes the construction of pig nuclear transfer embryos as an example.

[0038] 1. Isolation and culture of donor cells

[0039] The donor cells are from Guangdong Wenshi Group's premium boar ear tissue. After cutting the pig ear skin, put it in DMEM culture solution and store it at 4℃ and transport it back to the laboratory. After the pig ear skin tissue is cut into pieces, the tissue fragments are washed with DMEM Resuspend with appropriate amount of fetal bovine serum and transfer to a petri dish, 37℃, 5% CO 2 , Cultivate in saturated humidity environment. After 5-7h, add DMEM culture medium containing 10% fetal bovine serum, and pass the culture when the cells grow to 90% confluence. On the second day of passaging, change to cell culture medium (DMEM medium + 10% fetal bovine serum) containing ...

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Abstract

The invention relates to the biotechnology field, and in particular, relates to a method for improving the efficiency of porcine nuclear transfer. The method mainly includes that a BET protein inhibitor (+)-JQ1 is used for pre-treating donor cells before somatic cell nuclear transfer, and then the treated donor cells are used for constructing a porcine nuclear transfer embryo. The method is simpleand convenient, has obvious effect, can treat porous somatic cells at one time, can produce large-scale cloned embryos, and can significantly improve the in-vitro development efficiency of embryos cloned from the somatic cells.

Description

Technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving the efficiency of pig nuclear transplantation. Background technique [0002] Long before somatic cells were used as donors for cloned embryos, cloned animals with embryonic stem cells as donor cells were born, but embryonic stem cells are difficult to obtain and not easy to cultivate. For example, porcine embryonic stem cells have not been established successfully, so they cannot be widely used. Since 1997, Dolly Sheep has been widely used because of the use of somatic cells. Compared with stem cells, highly differentiated somatic cells have a unique transcriptome. This unique gene expression defines that somatic cells are not pluripotent. Therefore, it is easy to cause incomplete reprogramming during the cloning process, resulting in cloning efficiency low. [0003] Since the birth of the first somatic cloned mammal "Dolly" sheep in 1997, there have been reports of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0656C12N2501/999
Inventor 吴珍芳石俊松周荣贺晓燕许卫华罗绿花麦然标蔡更元
Owner WENS FOOD GRP CO LTD
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