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Cattle trophoderm stem cell system establishment method

A trophoblast stem cell and cell technology, which is applied in the field of cell biology and molecular biology, and can solve the problems of long-term passage and establishment of bovine TSC in vitro.

Active Publication Date: 2013-05-22
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, in terms of domestic animal TSC research, there are research reports on related culture systems abroad and some TSC cells have been obtained, but there is no report on the long-term passage and establishment of bovine TSC in vitro

Method used

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  • Cattle trophoderm stem cell system establishment method
  • Cattle trophoderm stem cell system establishment method
  • Cattle trophoderm stem cell system establishment method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 In vitro fertilization to obtain bovine blastocysts

[0044] Take fresh bovine ovaries from the slaughterhouse and wash them with sterilized physiological saline, extract the follicular fluid from the follicles with a syringe, add them to a sterile 10cm petri dish, use a suction pipette to pick the eggs under a stereo microscope and put them into bovine eggs Wash 3 times in the mature solution, transfer to the mature solution covered with 300 μl paraffin oil, and place at 38.5°C, 5% CO 2 After 22 hours of culture in the incubator, in vitro fertilization was carried out. Before in vitro fertilization, freshly prepare fertilization fluid A (BO fluid + caffeine) and B fluid (BSA + heparin) (A:B=1:1) (37°C), fertilization drop (20 μl / drop), development drop ( 40μl / drop). Take a sterile glass tube (with parafilm) and add 8ml of solution A. Take the frozen thin tube semen from liquid nitrogen and put it into a 37°C water bath to melt quickly, take out the thin tu...

Embodiment 2

[0045] Example 2 Preparation of Feeder Layer Mouse Fetal Fibroblasts+Wnt-3A Mouse Subcutaneous Connective Tissue Cells

[0046] The second generation of mouse fetal fibroblasts and Wnt-3A mouse subcutaneous connective tissue cells were mixed at a ratio of 1:1, and inoculated into a 10 cm cell culture dish at 38.5 ° C, 5% CO 2 Subcultured to the 4th passage, frozen. Press 1.1×10 after thawing 5 Each cell / well was inoculated in a four-well plate treated with 0.2% gelatin, and treated with 17 μg / ml mitomycin for 3 h when the confluence of the cells reached 60%-80%.

Embodiment 3

[0047] Example 3 Inoculation of blastocysts

[0048] Take the blastocyst on the 7th day and place it in 4mg / ml protease solution. When the zona pellucida becomes thinner, quickly put the blastocyst into the 2i small molecule inhibitor culture solution and wash twice, and then transfer it into the 2i small molecule inhibitor culture solution Blow lightly with a mouth pipette, and when the zona pellucida falls off, quickly put the blastocysts into the feeder layer freshly treated with mitomycin, and place them at 38.5°C, 5% CO 2 cultured in an incubator. On the 5th day, observe the adherence situation.

[0049] The formula of the 2i small molecule inhibitor culture solution is calculated in 1L as:

[0050]

[0051] Among them, PD0325901 is a non-ATP competitive MAPK kinase MEK inhibitor with molecular formula C 16 h 14 f 3 IN 2 O, the molecular structure is shown in formula (I):

[0052]

[0053] CHIR99021 is a GSK3β selective inhibitor with molecular formula C 22 ...

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Abstract

The invention provides a cattle trophoderm stem cell system establishment method which comprises the following steps: inoculating hemiblastula of which zona pellucida is removed through pronase treatment into a feed layer in mouse embryonic fibroblast subjected to mitomycin treatment and Wnt-3A mouse subcutaneous connective tissue cells, adding 2i small-molecule inhibitor culture solution to perform continuous cell culture of cells, and establishing a cattle trophoderm stem cell system. The method can be used for obtaining and culturing cattle trophoderm stem cells under in-vitro conditions for a long time. The cattle trophoderm stem cell system establishment method can be applied to large animal transgenosis and somatic cell cloning and can be further used for cattle embryo implantation and placenta differentiation.

Description

technical field [0001] The invention relates to the fields of cell biology and molecular biology, in particular to a method for establishing a line of bovine trophoblast stem cells. Background technique [0002] Trophoblast stem cells (TSCs) are precursor cells that differentiate into the placenta during embryonic development. In mice, TSC can be obtained after attachment of the trophectoderm (Trophectoderm, TE) at one end of the blastocyst. Compared with the pluripotency of embryonic stem cells (Embryonic stem cells, ESCs) derived from the inner cell mass (Inner cell mass, ICM) of blastocysts, the differentiation and development ability of TSCs is limited. TSC obtained in some species has been used to study cell differentiation (Flechon.J.E.1995; Talbot, 2000; Miyazaki, 2002; Hashizume K, 2006). [0003] The 2i system mainly includes CHIR99021 and PD0325901. CHIR99021 is a small molecule inhibitor targeting GSK3 (glycogen synthase kinase3) with well-defined characteristi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12R1/91
Inventor 李荣凤李雪玲黄翔华乌云毕力格
Owner INNER MONGOLIA UNIVERSITY
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