EXCISION OF SPECIFIC DNA-SEQUENCES FROM INTEGRATED RETROVIRAL VECTORSVIA SITE-SPECIFIC RECOMBINATION

Vectors for gene transfer and gene therapy were developed which combin e the advantages of the integrase and recombinase systems. This was ac hieved by inserting two loxP sites for specific DNA excision into an M ESV based retroviral vector. We show that this 'retroviral lex system' allows the infection of cells and the expression of transferred genes , In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excis ion from integrated retroviral lox vectors were performed by the use o f a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU sel ection for cells in which site-specific recombination has occurred res ults in large numbers of independent cell clones. These results were c onfirmed by detailed molecular analysis, In addition we developed retr oviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lex-sequences locat ed in the LTRs of retroviral vectors are stable during retroviral repl ication. Potential applications of this system would be the establishm ent of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.