Targeted recombination of exogenous DNA to its homologous chromosomal location is a potentially powerful tool for studying gene function, for creating animal models of disease, and for treating gene defects in humans. Unfortunately, targeted recombination in animal cells is masked by random integration into chromosomes, which occurs roughly 1000 times more frequently. As a consequence, gene-disruption and gene-replacement experiments of the sort that are commonplace in yeast are impractical in mammalian cells at present. For targeted approaches to contribute significantly to solving problems of gene function and disease, the proportion of targeted to random events must be increased. The objective of our proposed research is to understand the mechanisms of targeted and random recombination cells with a view toward increasing the ratio of targeted to random recombination events. We propose to develop a test system involving the APRT gene in its natural chromosomal context in CHO cells 1) to measure the relative proportions of targeted and random recombination into mammalian chromosomes and 2) to measure the relative frequencies of targeted integration and targeted gene conversions. Using this system, we will test two types of targeting substrates 1) to distinguish between break-repair models for targeted recombination in mammalian cells and 2) to determine which type of targeting substrate is more effective at promoting targeted recombination. In addition, we will test the effects of modifying the ends of targeting substrates in order to inhibit end-to-end ligation: 1) potentially increasing the fraction of substrates available for targeted recombination and 2) potentially decreasing their random integration into the chromosome. Finally, using a series of amplified mouse L cell lines, in which the ADA gene copy number ranges from 2 to 20,000 copies (all in the same chromosomal context), we will 1) measure the effect of target number on the frequency of targeted recombination and 2) screen for different efficiencies of targeted recombination at various sites within the amplified unit.
