Method for acquiring enucleated oocyte

A technology of oocytes and cumulus cells, applied in the field of obtaining enucleated oocytes, can solve the problems of PB1 deviation, lower enucleation rate, and impact on embryo quality

Inactive Publication Date: 2012-02-08
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This routine treatment can easily make PB1 deviate from the chromosome (plasma) of the egg, and the rate of enucleation decreases during the blind aspiration enucleat

Method used

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  • Method for acquiring enucleated oocyte

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Embodiment 1

[0026] Embodiment 1, somatic cell cloning

[0027] 1. Somatic cell culture and processing

[0028] 1. Isolation and culture of bovine adult fibroblasts

[0029] The isolated ear tip tissues of adult cattle (Chinese Herstein cattle) were obtained from Hushi Slaughterhouse. The ear skin is shaved with a scalpel first, and then disinfected with 70%-75% alcohol, and the ear tip is quickly cut off about 1cm with tissue scissors 3 Immediately immerse in 70%-75% alcohol for 60s, wash 2-3 times in sterilized PBS containing 250U / mL penicillin and 250μg / mL streptomycin, put into the same solution, and keep warm at 4℃. Back to the lab. In the ultra-clean workbench, the ear tissue was rinsed twice with PBS in a 60mm culture dish, then put into a penicillin bottle without adding any solution, and repeatedly cut the tissue piece to about 1mm with ophthalmic scissors 3 size. After washing 3 times with PBS and DMEM containing 20% ​​FBS (DMEM+20% FBS), pick the tissue pieces and inoculate...

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Abstract

The invention discloses a method for acquiring an enucleated oocyte. The invention also provides a method for acquiring an oocyte to be enucleated, comprising the steps of: 1) subjecting an in vitro cumulus-oocyte complex to in vitro maturation culture for 12-18h, and removing a cumulus cell so as to obtain a cumulus cell removed oocyte; 2) continuing conducting in vitro maturation culture to the cumulus cell removed oocyte obtained in step 1), thus obtaining an oocyte to be enucleated and with a first polar body. Experiments prove that in the steps of acquiring an enucleated oocyte, the two-step method provided in the invention can achieve a denucleation rate of 95%, thus maintaining the integrity of oocyte cytoplasm to the utmost.

Description

technical field [0001] The present invention relates to a method for obtaining enucleated oocytes. Background technique [0002] When performing somatic cell cloning, the routine treatment for oocytes before obtaining enucleated oocytes is: in vitro maturation (in vitro maturation, IVM) of cumulus oocytes extracted from the ovary and cultured for 18-20hr (bovine, sheep etc.), cumulus cells were removed after 40-44hr (pig). At this time, most of the oocytes have expelled the first polar body (the first polar body, PB1) and are in the metaphase of the second meiosis (metaphase II, MII). Oocytes with PB1 were then picked as recipient cytoplasm. This conventional treatment easily causes PB1 to deviate from the chromosome (plasma) of the egg, and the enucleation rate decreases during the blind aspiration enucleation process marked by PB1. However, the use of fluorescent dyes combined with ultraviolet irradiation can easily cause damage to eggs and affect the quality of embryos...

Claims

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Application Information

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IPC IPC(8): C12N5/075C12N15/877A01K67/027
Inventor 李光鹏
Owner INNER MONGOLIA UNIVERSITY
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