Efficient sequential gene regulation via FLP-and Cre-recombinase using adenovirus vector in mammalian cells including mouse ES cells.

Microbiol Immunol

Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Published: January 2007

AI Article Synopsis

  • Site-specific recombinase is an effective tool for gene regulation, but traditionally, it can only target one gene at a time.
  • The study demonstrates a method for sequentially regulating one or two genes using Cre-and FLP-expressing recombinant adenoviruses (rAd), optimizing dosages to minimize cytotoxicity.
  • This system is beneficial for gene function studies and could impact research on gene switching related to development and regeneration in mouse embryonic stem cells.

Article Abstract

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.

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http://dx.doi.org/10.1111/j.1348-0421.2006.tb03850.xDOI Listing

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