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Forward genetic screening for regulators involved in cholesterol synthesis using validation-based insertional mutagenesis.


ABSTRACT: Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s) due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM) system was used to isolate and characterize the 25-hydroxycholesterol (25-HC)-resistant and SR-12813-resistant mutants. Here we report that five mutant cell lines were isolated. Among which, four sterol-resistant mutants either contain a truncated NH2-terminal domain of sterol regulatory element-binding protein (SREBP)-2 terminating at amino acids (aa) 400, or harbor an overexpressed SREBP cleavage-activating protein (SCAP). Besides, one SR-12813 resistant mutant was identified to contain a truncated COOH-terminal catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). This study demonstrates that the VBIM system can be a powerful tool to screen novel regulatory genes in cholesterol biosynthesis.

SUBMITTER: Jiang W 

PROVIDER: S-EPMC4245081 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Forward genetic screening for regulators involved in cholesterol synthesis using validation-based insertional mutagenesis.

Jiang Wei W   Tang Jing-Jie JJ   Miao Hong-Hua HH   Qu Yu-Xiu YX   Qin Jie J   Xu Jie J   Yang Jinbo J   Li Bo-Liang BL   Song Bao-Liang BL  

PloS one 20141126 11


Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s) due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM) system was used to isolate and characterize the 25-hydroxycholesterol (25-HC)-resistant and SR-12813-resistant  ...[more]

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