Establishing method for parthenogenetic activation of source embryonic stem cell

An embryonic stem cell, parthenogenetic activation technology, applied in embryonic cells, germ cells, animal cells, etc., can solve the problems of easily damaged inner cell mass cells, unfavorable protective cells, decreased vitality, etc., to expand sources and research scope, reduce Exposure time, effect of protecting cells

Inactive Publication Date: 2009-05-13
王彦
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Problems solved by technology

[0009] The above literatures all use the immunosurgical method to separate the inner cell mass. The immune reaction is easy to damage the cells of the inner cell mass and reduce their vitality. It takes a long time and is not conducive to protecting cells, and the blastocyst rate is low.

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  • Establishing method for parthenogenetic activation of source embryonic stem cell
  • Establishing method for parthenogenetic activation of source embryonic stem cell
  • Establishing method for parthenogenetic activation of source embryonic stem cell

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Embodiment Construction

[0032] Taking Kunming mice as an example, the technical process for the establishment of the parthenogenetic activation-derived embryonic stem cell line of the present invention is: female Kunming mice hyperovulation induction - egg retrieval - application of calcium ionophore A23187 and 6-dimethylaminopurine (6-DMAP) to chemically activate it—mechanically separate inner cell mass cells—place inner cell mass cells in a culture dish with feeder cells laid out in advance—expand—passage—identify. Specific steps are as follows.

[0033] 1. Pregnant horse serum gonadotropin (PMSG) was used to act on female Kunming mice to perform superovulation induction, and the oocytes of female Kunming mice were as follows: figure 1 shown.

[0034] 2. After egg retrieval, apply calcium ionophore A23187 and 6-dimethylaminopurine (6-DMAP) to chemically activate them: digest with 80 μ / ml hyaluronidase for half a minute, then use human tubal fluid (human tubal fluid medium, HTF) was washed 3 times...

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Abstract

The invention provides a method for establishing a parthenogenetic activation derived embryonic stem cell system, which comprises the following steps: pregnant mare serum gonadotropin (PMSG) is used to act on a female animal body for superovulation; after ova are taken, a calcium ion carrier A23187 and 6-dimethylaminopurine (6-DMAP) are applied to perform chemical activation to the ova, and embryos developed by the activation are cultured in vitro to a blastula; and a mechanical method is used to separate cells of an inner cell mass, a thin needle is used to directly pierce the blastula of the embryos, and the inner cell mass is peeled and is subject to passage and amplification culture in a culture dish paved with feeder layer cells to establish a steady animal parthenogenetic activation ES cell system. The method has good effect of activation treatment, ensures that the blastula rate is up to 96.5 percent, avoids an immunologic reaction from damaging the cells of the inner cell mass by separating the cells of the inner cell mass with the piercing mechanical method, has short time consumption, reduces the exposure time of the cells outside an incubator, furthest protects the cells, and solves the immunogenicity problem in the application of embryo source, namely ES cells.

Description

technical field [0001] The invention relates to a method for establishing a parthenogenetic activation-derived embryonic stem cell line, and belongs to the technical field of embryonic stem cell lines. Background technique [0002] Embryonic stem cells (Embryonic stem cells), also known as ES cells, are obtained from the inner cell mass cells of blastocysts that develop for 5-7 days in vitro, and have the characteristics of unlimited proliferation and multi-directional differentiation potential. Human ES cells can be induced to differentiate into various human cells in vitro, and are widely used in clinical medicine and scientific research. However, because ES cells are derived from embryos, they are restricted by religion, morality and ethics in some countries and regions, which greatly limits the research and development of ES cells. [0003] Embryonic stem cells are currently divided into three types according to their sources: normal embryos (mostly), nuclear transfer a...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/0735
Inventor 王彦钱德俭陈子江宦晴韦多徐凯高选
Owner 王彦
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