Forward genetic screening for regulators involved in cholesterol synthesis using validation-based insertional mutagenesis
Autor: | Wei Jiang, Yu-Xiu Qu, Bao-Liang Song, Jing-Jie Tang, Hong-Hua Miao, Jie Qin, Jinbo Yang, Jie Xu, Bo-Liang Li |
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Rok vydání: | 2014 |
Předmět: |
Genetic Screens
Mutagenesis and Gene Deletion Techniques Mutant Genetic Vectors Gene Identification and Analysis Mutagenesis (molecular biology technique) lcsh:Medicine CHO Cells Biology Research and Analysis Methods Biochemistry Insertional mutagenesis Cricetulus Genetics Animals Humans Genetic Testing lcsh:Science Molecular Biology Techniques Gene Molecular Biology Regulator gene Multidisciplinary Diphosphonates lcsh:R Lentivirus Intracellular Signaling Peptides and Proteins Membrane Proteins Biology and Life Sciences Lipids Insertional Mutagenesis Forward genetics Hydroxycholesterols Sterol regulatory element-binding protein Protein Structure Tertiary Mutagenesis Insertional Cholesterol HEK293 Cells Gene Expression Regulation lcsh:Q lipids (amino acids peptides and proteins) Hydroxymethylglutaryl CoA Reductases Sterol regulatory element-binding protein 2 HeLa Cells Signal Transduction Sterol Regulatory Element Binding Protein 2 Research Article |
Zdroj: | PLoS ONE PLoS ONE, Vol 9, Iss 11, p e112632 (2014) |
ISSN: | 1932-6203 |
Popis: | 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-resisitant 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. |
Databáze: | OpenAIRE |
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