Rapid Generation of Isogenic Mammalian Cell Lines Expressing Recombinant Transgenes by Use of Cre Recombinase

Abstract

Mammalian cell lines expressing a recombinant gene of interest are conventionally generated by random genomic integration of exogenous DNA containing the recombinant gene and a selectable marker. In contrast, site-specific recombination targets the exogenous DNA to a preselected site in the mammalian genome (1,2). Genomic targeting with Cre recombinase of phage P1 is relatively simple and results in reproducible gene expression from a single copy of the inserted DNA (3). Thus, recombinase-mediated targeting can greatly speed the generation of stably transfected cell lines expressing a desired recombinant gene product. Because the cell lines obtained by Cre-mediated targeting are isogenic, one can eliminate the extensive functional screening required in conventional methods because of random integration and concomitant position and copy number effects on gene expression (4). Precise single-copy gene delivery to a defined chromosomal position is particularly valuable to characterize mutant regulatory sequences driving a reporter gene or to evaluate mutant alleles and variant isoforms of a particular recombinant gene, as has been done, for example, with the angiotensin AT₁ receptor (5).