Reporter gene testing method for enhancer and promoter functions of intron SNP of CYP3A4

A CYP3A4, reporter gene technology, applied in the field of molecular biology

Inactive Publication Date: 2014-01-22
ZHENGZHOU UNIV
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Problems solved by technology

So far, there is no ideal mammalian cell line with high expression of CYP3A4 enzyme in in vitro research, and the production of the commonly used liver cancer cell line HepG2 is extremely low. It is necessary to seek intronic enhancers or enhancers that can enhance the expression of CYP3A4 gene and increase the production of CYP3A4 enzyme. Intronic promoters with strong transcriptional activity will have important application value for in vitro research or screening of drugs metabolized by CYP3A4 enzymes, but in vitro methods to test that CYP3A4 intronic SNPs have both enhancer and promoter functions have not been reported yet

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  • Reporter gene testing method for enhancer and promoter functions of intron SNP of CYP3A4
  • Reporter gene testing method for enhancer and promoter functions of intron SNP of CYP3A4
  • Reporter gene testing method for enhancer and promoter functions of intron SNP of CYP3A4

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Embodiment Construction

[0063] specific implementation plan

[0064] Because the present invention relates to a human CYP3A4 intron sequence and a method for verifying that the SNP of the sequence has enhanced expression function, including selecting the intron sequence from the CYP3A4 genome, the conditions to be met by the CYP3A4 genome intron sequence are as follows:

[0065] 1.1 an intron located between the two exons of the CYP3A4 gene encoding the CYP3A4 enzyme, and

[0066] 1.2 The intron contains a typical GT...AG splice structure, that is, there is a GT splice site at the 5' end and an AG splice site at the 3' end, and

[0067] 1.3 Within 20 to 50 nucleotides upstream of the splicing junction at the 3′ end of the intron, there is another site that plays an important role in splicing, its sequence contains A, called the branch site, and

[0068] 1.4 In vivo experiments have confirmed that the SNP in this intron affects CYP3A4 enzyme activity, and

[0069] 1.5 The SNP variati...

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Abstract

The invention relates to a reporter gene method for an intron sequence of human CYP3A4 and for testing whether the SNP of the sequence has enhancer and promoter expression enhancing functions at the same time. The method comprises the following steps: selecting the intron sequence from the genome of human CYP3A4; constructing a luciferase reporter gene plasmid of a promoter sequence of CYP3A4; constructing a luciferase reporter gene vector of an intron SNP enhancer of CYP3A4; constructing a luciferase reporter gene vector of an intron SNP promoter of CYP3A4; culturing and transfecting HepG 2 cells; and determining the activity of dual-luciferase. With the method, it is discovered that the intron SNP of human CYP3A4 has enhancer and promoter functions at the same time, SNP capable of improving output of CYP3A4 enzyme can be screened by using the method, and the method has a critical application value in further in-vitro research on or screening of drugs used for metabolism of the CYP3A4 enzyme.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a report for verifying that the CYP3A4 intron SNP has enhancer and promoter functions for the human CYP3A4 intron sequence and verifying that the sequence SNP has enhancer and promoter enhanced expression functions at the same time genetic approach. Background of the invention [0002] CYP3A4 enzyme is the most important drug-metabolizing enzyme in the adult body, and the metabolized drugs account for about half of the drugs used in clinical practice. The activity of the enzyme affects the curative effect of the drug and is related to the adverse reaction of the drug, and there are obvious individual differences. Studies have shown that there are about 40-fold individual differences in the expression of CYP3A4 enzymes in the liver, and about 90% of this difference is caused by genetic variation in the human CYP3A4 gene. If the genetic markers that cause individual differences ca...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/66
Inventor 杨卫红张莉蓉杨鸽韩圣娜赵登职闫良王洋
Owner ZHENGZHOU UNIV
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