Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system.
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.
Coumoul
Xavier
X
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
W
Wang
Rui-Hong
RH
Deng
Chuxia
C
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D051034:Inhibitor of Apoptosis Proteins; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins; D034622:RNA Interference; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D000077022:Survivin; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D017468:Receptors, Fibroblast Growth Factor; D000077022:Survivin; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C496354:Fgfr2 protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; C045073:Cre recombinase; D019426:Integrases