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Method for constructing cell model and cell bank for chromosomal translocation by recombination of hTERT (human Telomerase Reverse Transcriptase)

A technology of chromosomal translocation and cell model, which is applied in the direction of recombinant DNA technology, cells modified by introducing foreign genetic material, libraries, etc., can solve problems such as inability to carry out research, and achieve the effect of normal differentiation and maintenance of chromosomes

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

[0009] However, there have been no literature reports on the construction of immortal cell models and cell banks using hTERT to study the pathogenesis of chromosomal translocations from the cellular level in vitro, and it is impossible to carry out research on related projects

Method used

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  • Method for constructing cell model and cell bank for chromosomal translocation by recombination of hTERT (human Telomerase Reverse Transcriptase)

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Embodiment Construction

[0015] 1. Extraction of hTERT: ① Digestion of pClneo-hTERT: hTERT is located between the EcoRI and SalI sites of the plasmid pClneo-hTERT, and the multiple cloning site (MCS) of the pLXSNneo vector contains EcoRI and XhoI restriction sites. Commercially purchased pCIneo-hTERT plasmid, dissolved in an appropriate amount of ultra-clean H 2 In O or TE buffer, add 2uL 10× digestion buffer and 18uL H 2 O, add restriction endonuclease EcoR I and Xho I 0.5ul each, 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) The reaction was terminated, and hTERT was amplified according to the conventional PCR method, and the amplified product was collected for electrophoresis. ②hTERT electrophoresis: Take electrophoresis grade agarose and use electrophoresis buffer to make 10% agarose gel, pour it into the sealed gel filling platform, insert the sample comb, and remove the sealing tape from the gel maki...

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Abstract

The invention relates to a method for constructing a cell model and cell bank for chromosomal translocation by recombination of hTERT (human Telomerase Reverse Transcriptase), and belongs to the field of medical science. The method is mainly characterized by comprising the following steps of carrying out dual-enzyme digestion on a plasmid pCIneo-hTERT and a vector pLXSNneo by virtue of endonucleases EcoR I and Xho I to obtain hTERT and pLXSNneo enzyme-digested products by linking through Ligation Mix, carrying out PCR amplification and separating through gel electrophoresis, constructing a pLXSNneo-hTERT recombinant, transforming the recombinant with DH5a competent cells, purifying, amplifying and picking ampicillin-resistant colonies, extracting a plasmid, transfecting with a lipidosome, carrying out in-vitro passage on chromosomal translocation cells in logarithmic growth so that the recombinants and cell DNA are integrated, carrying out enlarge culture, cloning a cell containing positive recombinants screened through G418, screening the cell of which a cell morphology, a growth curve, a chromosome karyotype, carcinogenicity tests of naked mice, the activity of telomerase in transfected cells, expression products of hTERT mRNA, immunohistochemical staining, a cell proliferate cycle and an apoptosis rate are in line with characteristics of an immortalized cell and are same as or similar to those of a primary cell as the hTERT-mediated cell model for chromosomal translocation, and cryopreserving in liquid nitrogen. The cell model lays a foundation for in-vitro research on the pathogenesis of chromosomal translocation in a cellular level in a long term.

Description

technical field [0001] The invention relates to hTERT (telomerase reverse transcriptase catalytic subunit) recombination (mediated) construction of a chromosomal translocation cell model and its cell bank, which is mainly used in the field of medical genetics to provide cell models for in vitro research on chromosomal translocation and Preserve 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 symptoms of chromosomal diseases, a...

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C40B50/06C40B40/02
Inventor 翁炳焕李晓马袁英马裕叶光勇金帆
Owner 翁炳焕
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