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Construction of chromosome translocation cell model and cell bank thereof by virtue of transfection of monkey kidney virus 40 capital T genes

A chromosomal translocation, monkey kidney virus technology, applied in the field of medical cytogenetics research, can solve problems such as the inability to carry out research projects

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

[0008] 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 caused by chromosomal translocation and environmental factors at the cellular level, and it is impossible to carry out related research projects

Method used

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  • Construction of chromosome translocation cell model and cell bank thereof by virtue of transfection of monkey kidney virus 40 capital T genes

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Embodiment Construction

[0014] 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 a chromosome translocation cell model and a cell bank thereof by virtue of transfection of monkey kidney virus 40 capital T genes, belonging to the filed of the field of medical researches. The construction is mainly characterized in that generally, T4DNA ligase, BamHI, pcDNA3.1 (-) DNA and SV40LTag DNA are used for constructing an SV40LTag-pcDNA3.1 (-) recombinant; the recombinant is purified by competent colon bacillus; and then is introduced to in-vitro passed or logarithmic-growing chromosome translocation tissue cell which is digested by collagenase II by using a lipofection transfection method, so that the recombinant and the DNA of the cell are integrated; cloned cells which are screened by G418 and contain positive recombinants are subjected to enlarging cultivation; the form, growth curve, and karyotype of the screened cells, growth of soft agar colonies, tumorigenesis test of a naked mouse, SV40 capital T gene detection in DNA of the transfected cells, mRNA expression product assay and DNA sequence determination results satisfy the characteristics of immortalized cells; the cells which are the same as or similar to primary cells are used as the SV40LT mediated chromosome translocation in-vitro study cell models and cryopreserved in liquid nitrogen, so that a foundation is laid for researching the pathogenesis of the chromosome translocation in vitro based on the cellular level for a long time.

Description

technical field [0001] The invention relates to the construction of a simian kidney virus 40 large T gene (SV40LT antigen gene or SV40LT) mediated (transfected) chromosomal translocation cell model and a cell bank thereof, and is mainly used in the field of medical cytogenetics research, and is a method for chromosomal translocation Research provides cell models and preserves its research resources. Background technique [0002] Chromosomal translocation is a kind of chromosomal aberration, which generally refers to the transfer of part or whole of one or several chromosomes to another or several chromosomes, or refers to the transfer of a part of one or several chromosomes to another one or several chromosomes. Parts of chromosomes are exchanged. It is usually divided into unbalanced translocations that lose genetic material and balanced translocations that do not lose genetic material. Individuals with unbalanced chromosomal translocations show corresponding clinical sym...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕黄荷凤徐晨明李晓赵永富金昱
Owner 翁炳焕
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