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Method for adipose-derived adult stem cell labeling by using exogenous green fluorescent protein (GFP)

A technology of green fluorescent protein and adult stem cells, which is applied in the fields of fluorescence/phosphorescence, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high price, low result, limited supply, etc. The effect of research cost reduction

Active Publication Date: 2008-09-24
SICHUAN UNIV
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Problems solved by technology

[0006] However, due to the very difficult conditions for the cultivation and reproduction of GFP transgenic mice, no such mice have been bred in China, and only a few laboratories abroad have the conditions for production and breeding, but the price is extremely expensive. The market price of transgenic mice is about 2,000 US dollars each, and the supply is extremely limited
Therefore, it cannot be applied to research on a large scale, which greatly hinders the extensive and in-depth research of adipose-derived stem cells in vivo and in vitro
Therefore, the problem of cell labeling of adipose stem cells has not been ideally solved, and it is also one of the main bottlenecks restricting the in-depth research of adipose stem cells.
Couffinhal (T. Couffinhal, Hum Gene Ther, 1997.8 (8): 929-934) found that the method of using Lac-Z as a reporter gene and X-gal as a chromogenic substrate to measure gene transfer efficiency would lead to seriously low results. The reason is that the activity of related enzyme proteins is limited by factors such as spatial structure and reaction conditions.

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  • Method for adipose-derived adult stem cell labeling by using exogenous green fluorescent protein (GFP)
  • Method for adipose-derived adult stem cell labeling by using exogenous green fluorescent protein (GFP)
  • Method for adipose-derived adult stem cell labeling by using exogenous green fluorescent protein (GFP)

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Embodiment

[0033] Main materials and reagents used in the embodiments of the present invention:

[0034] Materials used:

[0035] 4-6 weeks old Balb / c male mice (provided by the Experimental Animal Center of Sichuan University); green fluorescent protein transgenic mice (provided by the Laboratory of Ophthalmology and Genetic Diseases, West China Hospital, Sichuan University)

[0036] Reagents used:

[0037] α-MEM medium, trypsin-EDTA, HEPES (Gibco, USA);

[0038] Fetal bovine serum (FBS) (U.S. Hyclone Company);

[0039] Type I collagenase, bovine serum albumin (BSA), Krebs-Ringer's bicarbonate solution (KRB) ascorbic acid, insulin, TGF-β 1 (Sigma, USA);

[0040] Shuttle plasmid pAdTrack-CMV, virus backbone plasmid pAdEasy-1, Escherichia coli BJ5183 and DH5α, human embryonic kidney 293 cell line (provided by the Laboratory of Ophthalmology and Genetic Diseases, West China Hospital, Sichuan University);

[0041] Trypsin (Promeaga, USA);

[0042] Rabbit anti-mouse Aggrecan related ant...

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Abstract

The invention relates to a method for using an extrinsic green luminescence protein to label fat body stem cell. The method uses adenovirus vectors infection extrinsic green luminescence protein to get into the fat body stem cell of small mouse to displace original green luminescence protein GM small mouse fat body stem cell to label. It manifests that the Balb / c small mouse fat body stem cell of adenovirus vectors infection extrinsic green luminescence protein and the original green luminescence protein GM small mouse fat body stem cell have the same growth, value adding and diversity from morphology, mRNA and protein expressing level.

Description

1. Technical field: [0001] The present invention relates to a new method of using exogenous green fluorescent protein (GFP) to mark adipose body stem cells, more specifically, relates to a method of using exogenous green fluorescent protein (GFP) to transfect it into Balb / c In mouse adipose-derived stem cells, a new method for cell labeling in place of endogenous green fluorescent protein transgenic mouse adipose-derived stem cells. 2. Background technology: [0002] Stem cells can be divided into embryonic stem cells and adult stem cells according to their sources. The research on embryonic stem cells is not very optimistic due to the constraints of ethics, racial concepts and potential allogeneic rejection. [0003] Adult stem cells exist in mature tissues and organs of humans and mammals, have self-renewal and certain pluripotent differentiation potential, and are intermediate between totipotent cells and mature cells. Under natural conditions, adult stem cells tend to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N33/48C12Q1/00
Inventor 田卫东林云锋刘磊汤炜郑晓辉陈希哲刘家武肖金刚
Owner SICHUAN UNIV
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