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Serum-free separating and culturing method for sheep embryo stem cell

An embryonic stem cell and serum-free culture medium technology, applied in the biological field of embryonic stem cells, can solve the problems that restrict the research and application of ESC technology, and achieve the effect of highlighting technological progress and expanding research fields.

Inactive Publication Date: 2012-01-11
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

These have greatly restricted the research and application of ESC technology in sheep genetics and breeding.

Method used

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  • Serum-free separating and culturing method for sheep embryo stem cell
  • Serum-free separating and culturing method for sheep embryo stem cell
  • Serum-free separating and culturing method for sheep embryo stem cell

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Embodiment

[0043] Isolation and culture method steps: collect 42 blastocysts obtained from sheep cultured in vitro, use Tyrode'sSolution to remove the zona pellucida, inoculate the naked embryos in the sheep ESC serum-free culture medium provided by the invention, and use a 32-gauge needle to inoculate the naked embryos Embryos were fixed on the bottom of the culture dish, and at the same time, the inner cell mass of the blastocyst should be avoided as much as possible, and the nude embryo trophoblast cells should be stripped to the maximum extent, and then placed at 38.6 ° C, 5% CO 2 Cultivate under saturated humidity conditions, and the primary culture time is 7 days (see image 3 , 4 Primary colonies), and then subculture once every 4 days by a conventional mechanical method at a rate of 1:2-1:4, and half of the culture medium was replaced every 48 hours during the culture period. Routine alkaline phosphatase staining was performed on the obtained sheep ESCs of the 9th and 14th passa...

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Abstract

The invention provides a serum-free separating and culturing method for a sheep embryo stem cell, comprising the following steps of: removing a zona pellucida of sheep blastula cultured in vitro with a Tyrode's Solution and then inoculating the sheep blastula to a serum-free culture solution of the sheep embryo stem cell and fixing by using a needle head; placing under the conditions that the temperature is 38.6DEG C and the saturated humidity is 5 percent CO2 for culturing; during the culturing, replacing the culture solution in a half quantity every 48 hours with the pH value of 6.8-7.2 andprimarily culturing for 7-9 days; and carrying out the transfer culture once according to the ratio of 1:2-1:4 by adopting a conventional mechanical method, that is to say, transferring the sheep embryo stem cell cultured in vitro to the 16th generation. The serum-free culture solution of the sheep embryo stem cell is prepared from D-MEM / F-12+80ng / mL bFGF+3muMCHIR 99021+10mu L / mL N2+20mu L / mL B27+10mu L / mL NEAA+2mM L-Glutamine+0.2mM beta-Mercaptoethanol. Bared embryos are fixed at the bottom of a culture dish by using a No.32 needle head so as to avoid the damage to the cells in the blastula, and a baked embryo trophocyte is stripped off. The preferable inoculating amount of the baked embryos is 45-60 / groups. The method can ensure that the formation rate of primary colony of sheep embryo stem cells is improved to 33 percent (14 / 42).

Description

technical field [0001] The invention relates to the biotechnology of embryonic stem cells, adopts mechanical stripping and serum-free separation and culture technology, and provides a solid technical platform for the production of transgenic sheep. Background technique [0002] Embryonic stem cells (Embryonic stem cell, ESC) are obtained from the inner cell mass (ICM) of the blastocyst before implantation in mammals. Proliferative cell lines have specific biological characteristics that allow them to reintegrate with the ICM and participate in the entire process of embryonic development. Using ESC cloning technology, its integration efficiency is much higher than that of traditional nuclear transfer technology. It can produce more animals with genetic homogeneity in a short period of time, eliminating the need for descendant determination, and greatly improving the breeding efficiency of fine-bred livestock. At the same time, the use of ESC cells to produce transgenic lives...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735
Inventor 黄俊成赵云程汪立芹林嘉鹏陈童陈世彬
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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