Abstract
Gene targeting by single-stranded oligodeoxyribonucleotides (ssODNs) is a promising tool for site-specific gene modification in mouse embryonic stem cells (ESCs). We have developed an ESC line carrying a mutant EGFP reporter gene to monitor gene correction events shortly after exposure to ssODNs. We used this system to compare the appearance and fate of cells corrected by sense or anti-sense ssODNs. The slower appearance of green fluorescent cells with sense ssODNs as compared to anti-sense ssODNs is consistent with physical incorporation of the ssODN into the genome. Thus, the supremacy of anti-sense ssODNs, previously reported by others, may be an artefact of early readout of the EGFP reporter. Importantly, gene correction by unmodified ssODNs only mildly affected the viability of targeted cells and did not induce genomic DNA double-stranded breaks (DSBs). In contrast, ssODNs that were end-protected by phosphorothioate (PTO) linkages caused increased H2AX phosphorylation and impaired cell cycle progression in both corrected and non-corrected cells due to induction of genomic DSBs. Our results demonstrate that the use of unmodified rather than PTO end-protected ssODNs allows stable gene modification without compromising the genomic integrity of the cell, which is crucial for application of ssODN-mediated gene targeting in (embryonic) stem cells.