A method for improving the in vitro developmental ability of sheep oocytes

A technology of oocyte and in vitro development, applied in the field of histone deacetylation inhibitor trichostatin A, which can solve the problem of low maturation rate of negative oocytes

Active Publication Date: 2017-09-12
新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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Problems solved by technology

[0003] In a previous study of a method for screening and culturing sheep oocytes in vitro (patent number: ZL200910113567.9), it was found that the maturation rate of negative oocytes after BCB screening was significantly lower than that of positive oocytes. Therefore, How to improve the quality of negative oocytes has become an urgent problem to be solved

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  • A method for improving the in vitro developmental ability of sheep oocytes

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[0020] Example, the substandard BCB-oocytes obtained by staining and separation were washed 3 times with the mature culture medium, and put into the mature culture medium with a volume of 60 μL / drop balanced in the previous 2 hours at a rate of 28 pieces / drop. CO 2 box, at 5% CO 2 , 95% air conditions; take out the oocytes matured in vitro for 24 hours, treat them with 0.1% hyaluronidase for 45 seconds, blow off part of the cumulus cells; The density of 28 pieces / drop was added to the 60 μL in vitro fertilization droplet that had been balanced 2 hours before; the semen was thawed in a water bath, and transferred into a test tube containing in vitro fertilization fluid that had been balanced 2 hours before, and placed in CO 2 box, upstream 23min, take the supernatant, centrifuge at 1500rpm for 5min, discard the supernatant, and precipitate the remaining sperm, press 3×10 6 Add in vitro fertilization droplets at a density of 1 / mL, and incubate with the treated oocytes; take ou...

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Abstract

A method for improving the in vitro developmental ability of sheep oocytes provided by the present invention, the quality BCB-oocytes obtained by dyeing and separation are matured in vitro and fertilized in vitro with Qugu containing 200nMol / L histone deacetylation inhibitor Cultivate for 10 hours in the bacteriostatin A (TSA) medium, continue to cultivate with a medium that does not contain TSA, count the cleavage rate after 48 hours of culture, count the blastocyst rate after 7 days of culture, and separate the BCB+ oocytes obtained by staining Wash 2-3 times with mature culture medium, cultivate until 24h and use directly. This method has important practical value for the in vitro utilization of sheep oocytes.

Description

technical field [0001] The invention relates to a histone deacetylation inhibitor trichostatin A (TSA), a method for cultivating BCB negative oocytes with poor quality after BCB screening, which can significantly improve the in vitro embryonic development of sheep oocytes Rate. Background technique [0002] TSA is a non-competitive reversible histone acetylation inhibitor, which is derived from the metabolites of streptomycin, and its main function is to enhance the acetylation level of histones. TSA can specifically inhibit the activity of histone deacetylase, reduce the level of DNA methylation and activate the expression of imprinted genes and housekeeping genes. The high acetylation level of histones can enhance the interaction between transcription factors and nucleosomes, which is beneficial to the initiation of gene transcription. The embryonic genome is activated when histone acetylation peaks, coinciding with increased gene expression levels. Therefore, the histo...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075
Inventor 黄俊成汪立芹杨楠林嘉鹏吴阳升赵云程
Owner 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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