title: Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.
abstract: Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70-80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro.
authors.affiliation: Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA.
authors.forename: Ee Tsin
authors.initials: ET
authors.lastname: Wong
authors.forename: John L
authors.initials: JL
authors.lastname: Kolman
authors.forename: Yao-Cheng
authors.initials: YC
authors.lastname: Li
authors.forename: Larry D
authors.initials: LD
authors.lastname: Mesner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
authors.forename: Christian
authors.initials: C
authors.lastname: Berens
authors.forename: Geoffrey M
authors.initials: GM
authors.lastname: Wahl
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004318:Doxycycline / Q000494:pharmacology*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics; D006657:Histones / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D015533:Transcriptional Activation; D019076:Transgenes; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D006657:Histones; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases; D004318:Doxycycline