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Separation method and culture in vitro method for mouse female germline stem cells

A reproductive stem cell and culture method technology, which is applied in the field of separation and in vitro culture of mouse female reproductive stem cells, and achieves the effects of low requirement of experimental conditions, simple method and strong operability.

Inactive Publication Date: 2012-03-21
吴际
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the isolation and long-term culture of postnatal mammalian female reproductive stem cells has not been reported

Method used

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  • Separation method and culture in vitro method for mouse female germline stem cells
  • Separation method and culture in vitro method for mouse female germline stem cells
  • Separation method and culture in vitro method for mouse female germline stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Line establishment and transplantation of neonatal or adult mouse female germline stem cells

[0027] Step 1, Isolation of Female Reproductive Stem Cells

[0028] After the 5-day-old or 42-day-old female mice were sacrificed, the abdomen of the mice was wiped with 75% ethanol in a sterile ultra-clean bench, and then the abdomen was cut open to find the ovary along the oviduct. Gently cut off the ovaries, remove the fat and the end of the fallopian tubes in pre-cooled D-Hanks solution. Put the ovary into a 1.5ml ep tube filled with 100μl D-Hanks and cut it into pieces. Transfer the shredded tissue and liquid in the 1.5ml ep tube to a 12ml centrifuge tube, add 3.5ml D-Hanks containing 1mg / ml collagenase and 1ml D-Hanks solution, place in a 37°C water bath and gently Shake for 15 minutes. Then centrifuge at 1000rpm for 5min, wash with PBS buffer three times after centrifugation. Then immediately add 5ml of D-Hanks containing 0.5mg / ml trypsin, shake gently in the water ...

Embodiment 2

[0057] Construction of eGFP transgenic mice

[0058] Step 1, preparation of eGFP virus particles

[0059] (1) Preparation of plasmid

[0060] Take a sterilized EP tube, add 3 μg pMSCV-eGFP plasmid, 3 μg pVPack-GP (gag-pol-expressing vector) and 3 μg pVPack-VSV-G (env-expressing vector), then add 1ml absolute ethanol and 0.1 times volume of NaAc (3M). Mix with a pipette and place at -30°C for co-precipitation overnight. The mixed plasmids were centrifuged at 12,000 rpm at 4°C for 10 minutes, and the supernatant was discarded. Then add 1 ml of 70% ethanol, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant. The mixed plasmids were stored at 4°C overnight.

[0061] (2) Transfection of 293T cells

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Abstract

The invention provides a separation method for mouse female germline stem cells in the technical field of transgenosis engineering. The separation method comprises the following steps: step one, collecting a mouse ovary, and adopting a two-step enzymic digestion method for preparing germline stem cell suspension; and step two, adding mvh first antibody and then second antibody magnetic bead in the cell suspension and re-suspending the cells, thus separating the female germline stem cells. The invention also provides a culture in vitro method for the mouse female germline stem cells, which comprises the following steps: transferring the female germline cells on an STO cell culturing layer, adding female germline stem cell culture solution, and conducting primary culture and subculture on the female germline stem cells, thus obtaining purified and stable germline stem cells. In the method, the mouse female fermline stem cells are separated and cultured in vitro successfully at first time, the methods are simple, the cost is low, the requirements on experiment conditions are low, and the operability is strong.

Description

technical field [0001] The invention relates to an isolation method and an in vitro culture method of mouse female reproductive stem cells. Background technique [0002] Ovarian reproductive stem cells are the stem cells that maintain ovum production in the ovaries of female higher animals. In the classical theory of reproductive and developmental biology, the number of germ cells in the ovary of female mammals is fixed after birth, without the ability to proliferate, and the number of oocytes in the body decreases with age until menopause. At present, the research on male reproductive stem cells has been extensive in the world, and the techniques in isolation, identification, in vitro culture and in vivo transplantation have all become mature. [0003] Found through literature search to prior art, in 2004, American scientist Tilly published an article titled "Germ Line Stem Cells of Mammalian Ovary after Birth" in 2004 "Nature" magazine, volume 428, No. 6979, No. 6979, pag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
Inventor 吴际
Owner 吴际
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