Targeted recombination in Xenopus oocytes

The long-term goal of this project is to develop a technology that can produce genetic modifications at any position in the genome of a living cell. The central hypothesis is that the frequency of homologous recombination between an exogenous DNA and a reluctant target molecule, such as a chromosome, can be greatly enhanced by introducing a double-strand break (DSB) at the target site. The first part of the project demonstrated that a targeted DSB delivered in vivo by a site-specific endonuclease does indeed stimulate homologous recombination. The second part reported on attempts to create a more generalized in vivo, double-strand cleavage reagent. A psoralen-conjugated triplex-forming oligonucleotide was investigated for this purpose. Although psoralen is not a cleavage reagent, there were two reasons for choosing this molecule. The first was to aid in attempts to demonstrate triplex formation in Xenopus oocytes. The second was to investigate the ability of site-specific crosslinks to stimulate homologous recombination in oocytes. Unfortunately, no significant in vivo triplex formation or stimulation of recombination was observed in these experiments. These results contrast those of our collaborators, who were able to demonstrate both phenomena in COS cells using essentially the same reagents. These results suggest that a triplex based approach will be unsuccessful at enhancing the frequency of homologous recombination in oocytes, but may be useful in other cell types. Alternative approaches are discussed.