The Cre-loxP recombination system allows manipulation of DNA within a chromosomal environment. This system comprises a phage encoded recombinase (Cre) and a target site (loxP) in the genome. Recombination between two directly orientated loxP sites excises the intervening DNA as a circular molecular. Circular DNA sequences containing a single loxP site can be inserted as single copies into a pre-existing loxP site in the genome. The incorporation of loxP sites into retroviral vectors may offer several advantages. Firstly, it may allow removal of the potentially detrimental neomycin phosphotransferase gene (neoR gene) after selection of producer cell lines. Secondly, additional DNA sequences could be introduced into pre-existing proviral integrants of a high titer retroviral producer line. A retroviral vector containing the neoR gen and SV40 promoter and Herpes Simplex TK gene flanked by loxP sites has been constructed. High titer producer liens have been generated and shown to be G418 resistant and sensitive to ganciclovir. The ability of Cre recombinase to selectively remove the TK gene in these producer cell lines will be assessed. The titer of the TK-less producer cell line will be compared with the parental line. A second retroviral vector containing the neoR gene and the SV40 promoter with a single loxP site immediately 3' has also been constructed. High titer producer lies will be generated and the ability of Cre recombinase to insert a second selectable marker into the targeted site in the genome will be assessed. Once again the titer of this new producer line will be determined and compared to the parental line. This strategy may allow the generation of master producer lines in which targeted insertion of a gene of interest will rapidly generate high titer viral stocks.
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