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Cattle trophoderm stem cell system establishment method

A technology of trophoblast stem cells and stem cells, applied to embryonic cells, animal cells, vertebrate cells, etc., can solve the problems of long-term passage and line establishment of bovine TSCs that have not been seen in vitro

Active Publication Date: 2014-11-12
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 Bovine blastocysts obtained by in vitro fertilization

[0044] Take fresh cattle ovaries from the slaughterhouse and wash them with sterile saline. Use a syringe to extract the follicle fluid from the follicles and add them to a sterile 10cm petri dish. Use a mouth pipette to select the eggs under a stereo microscope and put them into the cow eggs Wash 3 times in the maturation liquid, transfer to the maturation liquid covered with 300μl paraffin oil, and place it at 38.5℃, 5% CO 2 After culturing in the incubator for 22 hours, in vitro fertilization was performed. Before in vitro fertilization, freshly prepared fertilization fluid A (BO fluid + caffeine) and B fluid (BSA + heparin) (A:B=1:1) (37℃), fertilization drops (20μl / drop), developmental drops ( 40μl / drop). Take a sterile glass tube (with parafilm) and add 8ml of A solution. Take a frozen thin tube of semen from liquid nitrogen and quickly melt it in a 37°C water bath. Take out the thin tube, wipe it wi...

Embodiment 2

[0045] Example 2 Preparation of feeder layer mouse fetal fibroblasts + Wnt-3A mouse subcutaneous connective tissue cells

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

Embodiment 3

[0047] Example 3 Inoculation of blastocysts

[0048] Take the 7th day blastocyst in 4mg / ml protease solution. When the zona pellucida is thinned, quickly put the blastocyst in 2i small molecule inhibitor culture solution and wash it twice, then transfer it into 2i small molecule inhibitor culture solution Blow gently with a mouth straw. When the zona pellucida has fallen off, quickly place the blastocysts in the feeder layer freshly treated with mitomycin, and place them at 38.5℃, 5% CO 2 In an incubator. Observe the adherence on the 5th day.

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

[0050]

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

[0052]

[0053] CHIR99021 is a selective inhibitor of GSK3β, molecular formula C 22 H 18 Cl 2 N 8 , The molecular structure is shown in formula (II):

[0054]

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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 present 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 (Trophoblast stem cells, TSC) are precursor cells that differentiate into the placenta during embryonic development. In mice, TSC can be obtained after adherence to the trophoblast (Trophectoderm, TE) at one end of the blastocyst. Compared with the pluripotency of embryonic stem cells (ESC) derived from the inner cell mass (ICM) of blastocysts, the differentiation and development ability of TSC is limited. TSC currently 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 high-efficiency and selective small molecule inhibitor targeting GSK3 (glycogen synthase kinase3) with clear ...

Claims

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Application Information

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Patent Type & Authority Patents(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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