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PMID: 15199177 Published · epublish English Journal Article

Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system.

Nucleic acids research ·Vol. 32 ·No. 10 ·2004-06-15 ·Pages e85

Coumoul X, Li W, Wang RH, Deng C

Abstract

RNA interference (RNAi) is a simple and powerful tool widely used for studying gene function in a number of species. Recently, inducible regulation of RNAi in mammalian cells using either tetracycline- or ecdysone-responsive systems has been developed to prevent potential lethality or non-physiological responses associated with persistent suppression of genes that are essential for cell survival or cell cycle progression. Here we show that the inducible regulation of RNAi also can be achieved by using a Cre-LoxP approach. We demonstrate that the insertion of a loxP-flanked neomycin cassette into RNA polymerase III promoter, which controls a vector-based RNAi unit, impairs the promoter activity. However, the expression of RNAi construct can be completely restored upon the removal of the neo cassette using a tamoxifen inducible Cre construct. We show that this system works with high efficiency in suppression of two endogenous genes, Fgfr2 and Survivin, in mouse embryonic stem (ES) cells, as evidenced by the decrease of levels of gene expression, reduced cell proliferation and colony formation. This system provides a potentially important yet simple approach to establish mutant mouse strains for functional study at defined stages upon turning on the inducible switches controlled by the Cre-LoxP system.

MeSH Terms
Animals Cell Line Cells, Cultured Embryo, Mammalian/cytology Genetic Vectors Green Fluorescent Proteins Inhibitor of Apoptosis Proteins Integrases/genetics,metabolism Kinetics Luminescent Proteins/genetics,metabolism Mice Microtubule-Associated Proteins/genetics,metabolism Neoplasm Proteins RNA Interference Receptor Protein-Tyrosine Kinases/genetics,metabolism Receptor, Fibroblast Growth Factor, Type 2 Receptors, Fibroblast Growth Factor/genetics,metabolism Stem Cells/cytology,metabolism Survivin Viral Proteins/genetics,metabolism
Chemicals
BIRC5 protein, human Inhibitor of Apoptosis Proteins Luminescent Proteins Microtubule-Associated Proteins Neoplasm Proteins Receptors, Fibroblast Growth Factor Survivin Viral Proteins Green Fluorescent Proteins Fgfr2 protein, mouse Receptor Protein-Tyrosine Kinases Receptor, Fibroblast Growth Factor, Type 2 Cre recombinase Integrases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Coumoul Xavier
Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Li Wenmei
Wang Rui-Hong
Deng Chuxia
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gnh083
Published
2004-06-15
Epub
2004-00-15
Pages
e85
Language
English
Region
England
NLM ID
0411011
PMCID
PMC434461
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/gnh083 PMC Full Text View journal details
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