SV40LT-mediated down syndrome cell model and construction method of cell bank thereof

A technology of Down syndrome and cell models, applied in the direction of cells modified by introducing foreign genetic material, chemical libraries, combinatorial chemistry, etc.

Inactive Publication Date: 2015-03-18
翁炳焕
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Problems solved by technology

[0010] In order to solve the problem of constructing an immortal cell model and its cell bank that can be used to study the pathogenesis of Down syndrome from the cellular level in vitro, the inventors proposed the present invention

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  • SV40LT-mediated down syndrome cell model and construction method of cell bank thereof

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Embodiment Construction

[0016] 1. Extraction of SV40 large T antigen DNA: ① SV40 DNA digestion: buy SV40 freeze-dried powder or SV40 plasmid containing large T antigen gene from the market, dissolve in appropriate amount of H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2 O, add restriction endonuclease BamH I (1-5U / ugDNA), incubate at 37°C for 1h, heat at 75°C for 15min, inactivate the enzyme, add 5uL electrophoresis loading buffer (also by adding 0.5mol / L EDTA ) to stop the reaction for electrophoresis. ②SV40 DNA electrophoresis: Take electrophoresis grade agarose and use electrophoresis buffer to make 10% agarose gel, pour it on the sealed gel filling platform, insert the sample comb, and remove the sealing tape from the gel making platform after the gel is solidified , pull out the comb, put it into the electrophoresis tank with enough electrophoresis buffer, the buffer is about 1mm above the surface of the gel, prepare the DNA sample with an appropriate amount of 10× loading buf...

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Abstract

The invention relates to an SV40LT antigen gene-mediated down syndrome cell model and construction method of a cell bank thereof applied to the field of medical research. The SV40LT antigen gene-mediated down syndrome cell model is mainly characterized by comprising the following processes: building an SV40LTag-pcDNA3.1(1) recombinant with T4DNA ligase, BamHI, pcDNA3.1(1) DNA and SV40LTag DNA according to a conventional method; purifying the recombinant with competent escherichia coli, introducing the recombinant into an in-vitro passage cell which is digested by collagenase II, or down syndrome histocyte which is in logarithmic growth by adopting a liposome transfection method, and merging the recombinant with DNA of the cell; carrying out enlarging cultivation, and screening cells containing positive recombinants to clone by G418; screening cell morphology, growth curve and karyotype; carrying out soft agar colony growth, nude mice tumor experiment, SV40 big T gene detection in transfection cell DNA, mRNA expression product determination and DNA sequence determination, wherein the result accords with the immortalized cell characteristics, and is the same as or similar to a primary cell to be cryopreserved in liquid nitrogen as an in-vitro research cell model for SV40LT-mediated down syndrome; and a foundation is laid for in-vitro long-term research of pathogenesis of the down syndrome from the cellular level.

Description

technical field [0001] The invention relates to a method for constructing a cell model of Down's syndrome mediated by simian kidney virus 40 large T antigen gene (SV40LT) and a cell bank thereof, which is mainly used in the field of reproductive health research, and provides and preserves the cell model for the research of Down's syndrome its research resources. Background technique [0002] Down's syndrome is also known as trisomy 21, that is, the patient has an extra chromosome 21, which is manifested as congenital stupidity. In my country, 16,000 to 20,000 cases of Down syndrome (trisomy 21 or congenital stupidity) are newly diagnosed every year, which will cause economic losses of 7.3 billion to 9 billion, and cause serious psychological burden and mental pain to society and families. , has seriously affected the population quality of our country, and has become a heavy burden on social development and a serious obstacle to sustainable development. [0003] In recent ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C40B50/06C04B40/02
Inventor 翁炳焕黄荷凤吕时铭杨红卫刘蓓
Owner 翁炳焕
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