DEVELOPMENT OF HIGH-TITER RETROVIRAL PRODUCER CELL-LINES BY USING CRE-MEDIATED RECOMBINATION

Retroviral gene transfer is widely used in experimental and human gene therapy applications. We have devised a novel method of generating hi gh-titer retroviral producer cell lines based on the P1 bacteriophage recombinase system Cre-loxP. Incorporation of loxP sites flanking a Ne o(r)-SVTK cassette in the proviral DNA allows excision of these select able markers through expression of Cre recombinase after production of a high-titer producer cell line. The resultant producer line contains a single loxP site Banked by the viral long terminal repeats. Retrans fection of this line with the Cre expression vector and a plasmid cont aining a gene of interest flanked by loxP sites allows insertional rec ombination of the gene into the favorable preexisting site in the geno me and the generation of a new line with a titer equivalent to that of the parental producer cell lint, The efficiency of the process is suf ficient to allow the generation of multiple new producer lines without the addition of antibiotic resistance genes. We have successfully gen erated retroviral vectors carrying different genes by using this appro ach and discuss the potential applications of this method in gene ther apy.