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A method for improving the in vitro developmental efficiency of nuclear transfer embryos

A nuclear transfer and embryonic body technology, applied in 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, low birth rate of cloned animals, etc. Effects of developmental efficiency, promotion of reprogramming, and inhibition of transcriptional memory

Active Publication Date: 2022-07-12
WENS FOODSTUFF GRP CO LTD
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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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  • A method for improving the in vitro developmental efficiency of nuclear transfer embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Nuclear transfer and fusion activation of reconstructed embryos

[0020] This example takes the construction of pig nuclear transfer embryos as an example:

[0021] 1. Donor cell isolation and culture

[0022] The donor cells were derived from the special-grade boar ear tissue of Guangdong Wen's Group. The pig ear skin was cut and placed in DMEM medium for storage at 4°C and transported back to the laboratory. After the pig ear skin tissue was cut into pieces, the tissue fragments were washed with DMEM. Resuspend with an appropriate amount of fetal bovine serum and transfer to a petri dish at 37°C, 5% CO 2 , Cultivated in a saturated humidity environment. After 5-7 hours, DMEM medium containing 10% fetal bovine serum was added, and the cells were passaged when they reached 90% confluence, and the somatic cells passed to the second passage were used as donor cells for nuclear transplantation.

[0023] 2. Oocyte collection and maturation culture

[0024] The...

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 medium and cultured for 168 hours.

[0030] Experimental group: The nuclear transfer embryos obtained after the operation in Example 1 were put into 5 groups of PZM-3 culture medium containing 1 nM, 10 nM, 15 nM, 20 nM and 50 nM (+)-JQ1 for 72 hours, respectively, and then transferred to different cultures. Continue to cultivate in PZM-3 medium containing (+)-JQ1 for 168h.

[0031] 2.2 In vitro culture of cloned embryos and blastocyst cell count

[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 the 7th day were fixed with 4%...

Embodiment 3

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

[0038] 3.1 Experimental Design

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

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

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

[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 the 7th day were fixed with 4% paraformaldehyde ...

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Abstract

The invention relates to the field of biotechnology, in particular to a method for improving the in vitro development efficiency of nuclear transfer embryos. The method for improving the in vitro development efficiency of nuclear transfer embryos provided by the present invention comprises: transferring the activated nuclear transfer reconstituted embryos into embryo culture medium containing BET protein inhibitor (+)-JQ1 for culturing. The method of the invention can inhibit cell-directed transcriptional memory, promote the reprogramming of nuclear transfer embryos, and thereby improve the in vitro development efficiency of somatic 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 cloned mammal "Dolly" sheep in 1997, there have been reports of the birth of cloned animals. However, the overall efficiency of animal cloning 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 into a pluripotent state. Studies have shown that compared with in vivo fertilized embryos, a large number of abnormal DNA and histone methylation and histone acetylation patterns appear in somatic cell nuclear transfer embryos. Epigenetic abnormalities can lead to abnormal gene expression in early embryonic developme...

Claims

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Application Information

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Patent Type & Authority Patents(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 FOODSTUFF GRP CO LTD
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