Development of a targeted flip-in system in avian DT40 cells.

Abstract

Gene-targeting to create null mutants or designed-point mutants is a powerful tool for the molecular dissection of complex phenotypes involving DNA repair, signal transduction, and metabolism. Because gene-targeting is critically impaired in mutants exhibiting attenuated homologous recombination (HR), it is believed that gene-targeting is mediated via homologous recombination, though the precise mechanism remains unknown. We explored gene-targeting in yeast and avian DT40 cells. In animal cells, gene-targeting is activated by DNA double strand breaks introduced into the genomic region where gene-targeting occurs. This is evidenced by the fact that introducing double strand breaks at targeted genome sequences via artificial endonucleases such as TALEN and CRISPR facilitates gene-targeting. We found that in fission yeast, Schizosaccharomyces pombe, gene-targeting was initiated from double strand breaks on both edges of the homologous arms in the targeting construct. Strikingly, we also found efficient gene-targeting initiated on the edges of homologous arms in avian DT40 cells, a unique animal cell line in which efficient gene-targeting has been demonstrated. It may be that yeast and DT40 cells share some mechanism in which unknown factors detect and recombine broken DNA ends at homologous arms accompanied by crossover. We found efficient targeted integration of gapped plasmids accompanied by crossover in the DT40 cells. To take advantage of this finding, we developed a targeted flip-in system for avian DT40 cells. This flip-in system enables the rapid generation of cells expressing tag-fused proteins and the stable expression of transgenes from OVA loci.