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
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
Externí odkaz: