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Construction of 18-trisomy syndrome cell model and cell bank of 18-trisomy syndrome cell by SV40LT gene transfection

A trisomy and cell model technology, applied to cells modified by introducing foreign genetic material, libraries, chemical libraries, etc., can solve problems such as inability to carry out research projects

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

[0009] However, there has been no literature report on the construction of an immortal cell model and its cell bank that can be used to study the pathogenesis of 18-trisomy at the cellular level in vitro, and it is impossible to carry out related research projects

Method used

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  • Construction of 18-trisomy syndrome cell model and cell bank of 18-trisomy syndrome cell by SV40LT gene transfection

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Experimental program
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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 (or 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 buffe...

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Abstract

The invention relates to construction of an 18-trisomy syndrome cell model and a cell bank of 18-trisomy syndrome cell by SV40LT gene transfection for the field of medical research. The construction is mainly characterized in that an SV40LTag-pcDNA3.1(-) recombinant is constructed through a T4DNA ligase, BamHI, pcDNA3.1(-) DNA and SV40LTag DNA as a matter of routine, the recombinant is purified through competent escherichia coli, the recombinant is introduced into an in-vitro passage cell digested through collagenase II or an 18-trisomy syndrome tissue cell which is logarithmically grown through a lipoid transfection method to be integrated with cell DNA, and a cloned cell which is screened through G418 and contains a positive recombinant is subjected to amplification culture; and a cell which accords with the characteristics of an immortalized cell and is the same as or similar to a primary cell on a cell morphology, a growth curve, a chromosome karyotype, soft agar colony growth, a nude mouse tumorigenesis test, SV40 large T gene detection of transfection cell DNA, an mRNA expression product measurement result and a DNA sequence measurement result is screened as the 18-trisomy syndrome in-vitro research cell model and cryopreserved in liquid nitrogen, so that the foundation is laid for researching the pathogenesis of 18-trisomy syndrome for a long time from cell level in vitro.

Description

technical field [0001] The invention relates to the transfection (mediated) 18-trisomy cell model and the construction of the cell bank of the simian kidney virus 40 large T antigen gene (SV40LT gene), which is mainly used in the field of reproductive health research, and is 18-trisomy synthesis It provides cell models and preserves its scientific research resources. Background technique [0002] Trisomy 18 is one of the major birth defects, also known as Edward's syndrome, that is, the patient has an extra chromosome 18. The main symptoms of this syndrome are kyphosis, narrow eye cleft, and ear Deformity, low ear position, small jaw, short sternum, etc. Due to severe mental retardation and multiple deformities in children, 50% died within 2 months after birth, less than 10% lived to 1 year, and very few could live to 15 years old. Due to the lack of effective treatment methods, once a child is born, it will bring a great mental and economic burden to the family and societ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B40/02C40B50/06
Inventor 翁炳焕黄荷凤陈松长李晓刘蓓陈雁
Owner 翁炳焕
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