Simultaneous Cre-mediated conditional knockdown of two genes in mice.
One of the most promising techniques to manipulate gene expression in vivo is the use of RNA interference (RNAi). Various approaches were developed to use RNAi in the mouse, including vector-based expression of short hairpin RNAs and the Cre/loxP recombination system. We combined these two approaches to create a vector system that allows the time- and tissue-specific control of two genes at the same time. For this purpose two independent conditional shRNA expression cassettes are combined into a single construct. By the use of different, incompatible pairs of lox sites that flank transcriptional stop cassettes, Cre recombinase can independently activate both short hairpins. Here, we show that Cre simultaneously activates both shRNAs in vitro and in vivo. We applied this technique to silence the widely coexpressed gene pairs Gsk-3alpha/Gsk-3beta and Erk1/Erk2 in murine embryonic stem cells and in the mouse brain.
Steuber-Buchberger
Patricia
P
Institute for Developmental Genetics, GSF National Research Center for Environment and Health, Ingolstädter Landstr.1, Munich/Neuherberg, Germany.
Wurst
Wolfgang
W
Kühn
Ralf
R
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018390:Gene Targeting / Q000379:methods*; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics* / Q000378:metabolism; D019426:Integrases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
D007527:Isoenzymes; D034741:RNA, Small Interfering; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D038362:Glycogen Synthase Kinase 3; C045073:Cre recombinase; D019426:Integrases