Cell test system and construction method and use

A cell and system technology, applied in the fields of cell biology and molecular biology, to achieve the effects of convenient detection, reduced research and development costs, and high sensitivity

Inactive Publication Date: 2003-04-30
HUADA GENE RES & DEV CENT HANGZHOU
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Problems solved by technology

The half-life of d2EGFP is only 2 hours

Method used

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  • Cell test system and construction method and use
  • Cell test system and construction method and use

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Embodiment Construction

[0018] We first constructed the 4xCRE-d2EGFP plasmid (4x is repeated 4 times), and cloned 4xCRE together with the HSV-tk promoter into the reporter gene d2EGFP. HSV-tk (Herpes simplex virus thymidinekinase) is herpes simplex virus thymidine kinase. The constructed plasmids were transformed into CHO-K1 and HEK-293 cell lines, screened with G418, clones were selected for identification, and positive clones were stimulated with foscolin (foscolin is a plant extract that has the effect of increasing the concentration of cAMP in cells) , 5 hours later, green fluorescence can be seen under the fluorescence microscope.

[0019] We have successfully constructed a reporter gene cell test system for drug screening and research. These two test systems can be used to detect changes in the concentration of cAMP in cells. All GPCRs (G protein coupled receptors) based on the cAMP signal transduction pathway can use this model to screen agonists and antagonists. . From the compounds screened out,...

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Abstract

A cell test system is the CHO-K1/d2EGFP and HEK-293/d2EGFP lines, which contains 4xCPE and the DNA sequence of HSV-TK. A method for configuring said cell test system includes such steps as designing 4xCRE-HSV-TK sequence, artificially synthesizing relative oligonucleotide, designing primer, PCR amplification, cloning it to d2EGFP carrier, sequencing the linked resultant, choosing correct recombinant plasmid, tansferring it in CHO-K1 and HEK-293 cells, screening resistant clone and function test. Said cell test system can be used to screen the compound mutually acting on CAMP signal transmission path.

Description

Technical field [0001] The present invention relates to the fields of cell biology and molecular biology, in particular to a cell test system and its construction method and application. Background technique [0002] In these two cell models, we used a reporter gene called d2EGFP. GFP (greenfluorescent protein) is a gene reporter molecule derived from jellyfish. It can be used to detect gene expression and protein location in vivo, in situ, and instantaneously. No additional protein or substrate is required. GFP will emit bright light under blue light excitation. Green fluorescence. GFP fluorescence is stable, does not depend on species, and has no damage to living cells. The improved GFP (enhanced green fluorescent protein) produces about 35 times stronger fluorescence than wild-type GFP. The codons favored by humans are used to increase the expression level. d2EGFP is modified on the basis of EGFP. EGFP is fused with the 422-461 amino acid residues of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/00C12Q1/68
Inventor 阎东升董伟谈学海胡咏武汪建
Owner HUADA GENE RES & DEV CENT HANGZHOU
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