title: Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells.
abstract: Stable transformants of mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We have eliminated both of these constraints on predictable gene expression by use of a lox recombination vector. The positive selection vector system is designed to directly select Cre-mediated DNA integration at a lox target previously placed into the genome of cultured mammalian cells. Proper targeting activates expression of a defective lox-neomycin phosphotransferase (neo) fusion gene target. With CHO cell lines containing this target, almost all of the selected transformants (54 of 56 independent G418-resistant colonies) were simple single-copy integrants of the targeting DNA. To monitor gene expression at a single chromosomal site, we used a beta-actin promoter-lacZ reporter construct. Independent G418-resistant colonies from site-specific integration of the reporter gene all showed nearly identical levels of beta-galactosidase activity when the reporter construct integrated at a particular chromosomal position. The same construct integrated at a second chromosomal position exhibited a slightly different level of activity, characteristic of that second position. These results show that Cre-mediated site-specific integration can facilitate the construction of isogenic cell lines and thereby permit reproducible gene expression in stably transformed cell lines.
authors.affiliation: DuPont-Merck Pharmaceutical Co., Wilmington, DE 19880-0328.
authors.forename: S
authors.initials: S
authors.lastname: Fukushige
authors.forename: B
authors.initials: B
authors.lastname: Sauer
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D066298:In Vitro Techniques; D019426:Integrases; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection; D014764:Viral Proteins
chemical_list: D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases