Parameters of oligonucleotide-mediated gene modification in mouse ES cells

Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. Here, we have studied the role of ssODN composition, transcription and replication of the target locus, and DNA repair pathways to gain more insight into the parameters governing ssODN-mediated gene targeting in mouse ES cells. We demonstrated that unmodified ssODNs of 35-40 nt were most efficient in correcting a chromosomally integrated mutant neomycin reporter gene. Addition of chemical modifications did not further enhance the efficacy of these ssODNs. The observed strand bias was not affected by transcriptional activity and may rather be caused by the different accessibility of the DNA strands during DNA replication. Consistently, targeting frequencies were enhanced when cells were treated with hydroxyurea to reduce the rate of replication fork progression. Transient down-regulation of various DNA repair genes by RNAi had no effect on the targeting frequency. Taken together, our data suggest that ssODN-mediated gene targeting occurs within the context of a replication fork. This implies that any given genomic sequence, irrespective of transcriptional status, should be amenable to ssODN-mediated gene targeting. The ability of ES cells to differentiate into various cell types after ssODN-mediated gene targeting may offer opportunities for future therapeutic applications.