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A method for improving the efficiency of pig 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 development efficiency of cloned embryos, excessive methylation of embryos, and low expression level, so as to improve the efficiency of in vitro development, increase the efficiency of cloning, Obvious effect

Active Publication Date: 2022-06-07
WENS FOODSTUFF GRP CO LTD
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  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A method for improving the efficiency of pig nuclear transfer
  • A method for improving the efficiency of pig nuclear transfer
  • A method for improving the efficiency of pig nuclear transfer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 (+)-JQ1 different concentrations on the impact of improving nuclear transfer efficiency

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

[0024] 1. Isolation and culture of donor cells

[0025] The donor cells come from the super boar ear tissue of Guangdong Wenshi Group. The pig ear skin is cut and stored in DMEM culture medium at 4°C and transported back to the laboratory. After the pig ear skin tissue pieces are cut into pieces, the tissue fragments are washed with DMEM Resuspend with appropriate amount of fetal bovine serum and transfer to culture dish, 37°C, 5% CO 2 , cultured in a saturated humidity environment. After 5-7 hours, add DMEM medium containing 10% fetal bovine serum, and subculture when the cells grow to 90% confluence. On the second day of passaging, change to cell culture medium (DMEM medium + 10% fetal calf serum) containing different concentrations of (+)-JQ1, treat for 72 hours, and change the medium every 24 ...

Embodiment 2

[0036] Example 2 Influence of different time on the efficiency of nuclear transfer by culturing donor cells with culture medium containing (+)-JQ1

[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 come from the super boar ear tissue of Guangdong Wenshi Group. The pig ear skin is cut and stored in DMEM culture medium at 4°C and transported back to the laboratory. After the pig ear skin tissue pieces are cut into pieces, the tissue fragments are washed with DMEM Resuspend with appropriate amount of fetal bovine serum and transfer to culture dish, 37°C, 5% CO 2 , cultured in a saturated humidity environment. After 5-7 hours, add DMEM medium containing 10% fetal bovine serum, and subculture when the cells grow to 90% confluence. On the second day after subculture, replace with cell culture medium (DMEM medium + 10% fetal calf serum) containing (+)-JQ1 wit...

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Abstract

The invention relates to the field of biotechnology, in particular to a method for improving the efficiency of nuclear transfer in pigs. The method mainly includes using BET protein inhibitor (+)-JQ1 to pretreat donor cells before somatic cell nuclear transfer, and then using the treated Donor cells for porcine nuclear transfer embryo construction. The method of the invention is simple and effective, and the porous somatic cells can be processed once to produce large-scale cloned embryos, and the in vitro development efficiency of somatic cell cloned embryos can be significantly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving the efficiency of nuclear transplantation in pigs. Background technique [0002] Long before somatic cells were used as cloned embryo donors, embryonic stem cells were used as donor cells to clone animals, but embryonic stem cells were difficult to obtain and difficult to cultivate. For example, pig embryonic stem cells have not yet been successfully established, so they cannot be widely used. Since 1997, Dolly the sheep has made cloning technology widely available due to 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 do not possess pluripotency, so it is easy to cause incomplete reprogramming during the cloning process, resulting in cloning efficiency. low. [0003] Since the birth of the first somatic cell cloned mammal "Dolly" sheep ...

Claims

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Application Information

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Patent Type & Authority Patents(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 FOODSTUFF GRP CO LTD
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