9529071 Crain The goal of this project is to develop a new and fairly simple method for creating targeted point mutations in genes in mice. Should the approach prove successful it would substantially increase our ability to create precise modifications in targeted genes in vivo. This would make it possible to carry out much more sophisticated analyses of mammalian gene function and regulation, and thus greatly enhance the study of mammalian genetics and development. The experimental strategy is to promote homologous recombination with targeted genes in embryos by injecting single strand oligonucleotides (carrying the desired sequence change) into pronuclei. The theoretical basis for this approach is that triple stranded DNA structures appear to be required intermediates of normal homologous recombination. The empirical basis is that it has been shown that introduced single strand DNA and oligonucleotides participate in homologous recombination in mammalian cells and yeast, indicating that they probably are incorporated into the putative triple strand intermediates. Also, it has been suggested that single strand DNA is less likely to incorporate randomly into host genomic DNA than is double strand DNA. If this is the case, then the upper limit of the amount of DNA that can be introduced without nonspecific toxicity to the embryos should be much higher with single than with double strand molecules. The critical question that will be tested in these experiments is whether a sufficiently high number of single strand molecules can be injected to raise the rate of homologous recombination with targeted genes to a level that can be detected without genetic selection. ***
