Highly efficient generation of signal transduction knockout mutants using a fungal strain deficient in the mammalian ku70 ortholog

摘要: Abstract Targeted gene replacement via homologous recombination is a routinely used approach to elucidate the function of unknown genes. Integration exogenous DNA in genomic requires action double-strand repair mechanisms. The filamentous ascomycete Sordaria macrospora model system for studying fruiting body development fungi. In contrast budding yeast Saccharomyces cerevisiae, but similar many fungi, plants and animals, transformed ectopically integrated into genome S. macrospora. Most probably this occurs by non-homologous end joining (NHEJ), mechanism that involves binding Ku heterodimer (Ku70/Ku80) at ends break (DSB). Phylogenetic analysis Ku70 orthologs fungal, plant, animal origin reveals protein well conserved among eukaryotes. To improve targeting efficiency macrospora, we identified deleted ku70 gene. No impairment Δku70 mutant vegetative or nor ascospore was observed making strain an ideal recipient developmental As case study, targeted deletion pheromone ppg2 receptor pre2. PCR generated constructs containing 1000 bp flanking sequence resulted drastically increased efficiency. consequence, almost all transformants carried disrupted target