HIGH-FREQUENCY ILLEGITIMATE INTEGRATION OF TRANSFECTED DNA AT PREINTEGRATED TARGET SITES IN A MAMMALIAN GENOME

To examine the mechanisms of recombination governing the illegitimate integration of transfected DNA into a mammalian genome, we developed a cell system that selects for integration events in defined genomic re gions. Cell lines with chromosomal copies of the 3' portion of the ade nine phosphoribosyltransferase (APRT) gene (targets) were established. The 5' portion of the APRT gene, which has no homology to the integra ted 3' portion, was then electroporated into the target cell lines, an d selection for APRT gene function was applied. The reconstruction of the APRT gene was detected at frequencies ranging from less than 10(-7 ) to 10(-6) per electroporated cell. Twenty-seven junction sequences b etween the integrated 5' APRT and its chromosomal target were analyzed . They were found to be randomly distributed in a 2-kb region immediat ely in front of the 3' portion of the APRT gene. The junctions fell in to two main classes: those with short homologies (microhomologies) and those with inserted DNA of uncertain origin. Three long inserts mere shown to preexist elsewhere in the genome. Reconstructed cell lines we re analyzed for rearrangements at the target site by Southern blotting ; a variety of simple and complex rearrangements were detected. Simila r analysis of individual clones of the parental cell lines revealed an alogous types of rearrangement, indicating that the target sites are u nstable. Given the high frequency of integration events at these sites , we speculate that transfected DNA may preferentially integrate at un stable mammalian loci. The results are discussed in relation to possib le mechanisms of DNA integration.