Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference.
RNA interference (RNAi)-mediated gene knockdown is a potent approach for studying gene function. We have previously reported a plasmid-based, tamoxifen-inducible gene knockdown system in cultured cells using a combined RNAi and Cre-LoxP system. Here, we validate this system in mouse and show that it can be used to suppress the expression of an endogenous gene (Fgfr2) with high efficiency. We show that transgenic mice carrying the U6-ploxPneo-Fgfr2 RNAi construct are normal, displaying Fgfr2 transcripts equivalent to those of wild-type controls, indicating that the U6 promoter is inactive in vivo due to the presence of the neo in the promoter. After excision of the neo by crossing with transgenic mice that express Cre in the mouse germline, the U6 promoter is activated, leading to over 95% reduction of Fgfr2 transcripts, and consequently, embryonic lethality. On the other hand, activation of the U6 promoter using transgenic mice that express Cre in the progress zone of the limb results in live mice with malformation of digits of both the forelimbs and hindlimbs. This method provides a fast, yet efficient way to decipher gene functions in vivo in a tissue-specific manner.
Coumoul
Xavier
X
Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health Bethesda, Maryland, MD 20892, USA.
Shukla
Vivek
V
Li
Cuiling
C
Wang
Rui-Hong
RH
Deng
Chu-Xia
CX
D000818:Animals; D005804:Genes, Lethal; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D017880:Limb Deformities, Congenital / Q000209:etiology; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics*; D034622:RNA Interference; D012320:RNA Polymerase III / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017468:Receptors, Fibroblast Growth Factor; D014764:Viral Proteins; C496354:Fgfr2 protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; C045073:Cre recombinase; D019426:Integrases; D012320:RNA Polymerase III