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Method for improving in-vitro development efficiency of nuclear transfer embryos

A technology of nuclear transfer and embryoid bodies, applied to embryonic cells, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problems of low survival rate of cloned animals and low birth rate of cloned animals

Active Publication Date: 2018-11-30
WENS FOOD GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Epigenetic abnormalities can lead to abnormal gene expression in early embryonic development, resulting in a lower birth rate of cloned animals, and more deformities such as large tongues, splayed feet, etc., resulting in a lower survival rate of cloned animals

Method used

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  • Method for improving in-vitro development efficiency of nuclear transfer embryos

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Nuclear transfer and reconstituted embryo fusion activation

[0020] This embodiment takes pig nuclear transfer embryo construction as an example:

[0021] 1. Isolation and culture of donor cells

[0022] 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 culture medium containing 10% fetal bovine serum, and subculture when the cells grow to 90% confluence, and use the somatic cells passed to the second generation as donor cells for nuclear transfer.

[0023] 2. Oocyte collection and maturation culture

[0024] The ovaries obtained from sows in the slaughte...

Embodiment 2

[0027] Example 2 Effects of different concentrations of (+)-JQ1 on the in vitro development of nuclear transfer embryos

[0028] 2.1 Experimental design

[0029] Control group: the nuclear transfer embryos obtained after the operation in Example 1 were placed in PZM-3 culture medium and cultured for 168 hours.

[0030] Experimental group: the nuclear transfer embryos obtained after the operation in Example 1 were respectively placed in five groups of PZM-3 culture solutions containing 1nM, 10nM, 15nM, 20nM and 50nM (+)-JQ1 and cultured for 72h, and then transferred to different Continue to culture in PZM-3 medium containing (+)-JQ1 until 168h.

[0031] 2.2 In vitro culture of cloned embryos and counting of blastocyst cells

[0032] The cloned embryos were cultured at 39°C, 5% O 2 , 5%CO 2 , 90% N 2 and saturated humidity. The cleavage and blastocyst development were observed and recorded on the 2nd and 7th day of culture respectively. The blastocysts on day 7 were fixed...

Embodiment 3

[0037] Example 3 Effects of different treatment times of (+)-JQ1 on the in vitro development of nuclear transfer embryos

[0038] 3.1 Experimental design

[0039] Control group (same as in Example 2): the nuclear transfer embryos obtained after the operation in Example 1 were placed in PZM-3 culture medium and cultured for 168 hours.

[0040] Experimental group: the nuclear transfer embryos obtained by the operation in Example 1 were placed in the PZM-3 culture medium containing 10nM (+)-JQ1 and cultured for 24h, 48h, 72h and 120h respectively, and then transferred to the culture medium without (+)-JQ1 Continue culturing in PZM-3 medium until 168h.

[0041] 7.2 In vitro culture of cloned embryos and blastocyst cell counting

[0042] The cloned embryos were cultured at 39°C, 5% O 2 , 5%CO 2 , 90% N 2 and saturated humidity. The cleavage and blastocyst development were observed and recorded on the 2nd and 7th day of culture respectively. The blastocysts on day 7 were fixe...

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Abstract

The invention relates to the biotechnology field, and in particular, relates to a method for improving the in-vitro development efficiency of nuclear transfer embryos. The method for improving the in-vitro development efficiency of the nuclear transfer embryos includes that activated nuclear transfer reconstructed embryos are transferred into an embryo culture liquid containing a BET protein inhibitor (+)-JQ1 and are cultured. The method can inhibit directional transcriptional memory of cells, promotes reprogramming of the nuclear transfer embryos, thereby improving the in-vitro development efficiency of the cell nuclear transfer embryos.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving the in vitro development efficiency of nuclear transfer embryos. Background technique [0002] Since the birth of the first somatic cell cloned mammal "Dolly" sheep in 1997, there have been reports of the birth of cloned animals. However, the overall efficiency of cloning animals is still low. [0003] In all mammals produced by somatic cell nuclear transfer (SCNT), defective epigenetic reprogramming may be the main reason for the low success rate. Histone modifications and DNA methylation are major factors hindering the reprogramming of somatic cells to a pluripotent state. Studies have shown that a large number of abnormal DNA and histone methylation and histone acetylation patterns appear in somatic cell nuclear transfer embryos compared with in vivo fertilized embryos. Epigenetic abnormalities can lead to abnormal gene expression in early embryonic develo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0604C12N2510/00C12N2501/999
Inventor 吴珍芳石俊松许卫华周荣罗绿花麦然标蔡更元
Owner WENS FOOD GRP CO LTD
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