Efficient sequential gene regulation via FLP-and Cre-recombinase using adenovirus vector in mammalian cells including mouse ES cells.
Site-specific recombinase is widely applied for the regulation of gene expression because its regulatory action is strict and efficient. However, each system can mediate regulation of only one gene at a time. Here, we demonstrate efficient "sequential" gene regulation using Cre-and FLP-expressing recombinant adenovirus (rAd) in two different monitor cell lines, for regulation of one gene (OFF-ON-OFF) and for two genes (ON-OFF and OFF-ON, independently). Generally, serial use of Cre-and FLP-expressing rAd tends to cause significant cytotoxicity, but we here described optimum dose of the rAds for serial regulation. We also established an efficient method of rAd infection to mouse ES cell lines after removing feeder cells, showing that this system is useful for removal of FRT-flanked drug-resistance gene cassette from recombinant ES cells prior to introduction of ES cells into blastocytes for chimeric mice production. Because our sequential gene-regulation system offers efficient purpose-gene regulation and strict OFF-regulation, it is potentially valuable for elucidating not only novel gene functions using cDNA microarray analysis but also for "gene switching" in development and regeneration research.
Kondo
Saki
S
Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Takahashi
Yuzuka
Y
Shiozawa
Seiji
S
Ichise
Hirotake
H
Yoshida
Nobuaki
N
Kanegae
Yumi
Y
Saito
Izumu
I
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000502:physiology*; D051379:Mice; D011995:Recombination, Genetic
C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases