new strategy for comprehensive analysis of gene functions in embryonic stem cells at present the limitation of phenotypebased genetic screening in embryonic stem cells escs is the diploid nature of the genome since it is known that cells deficient in the blooms syndrome gene blm show an increased rate of homologous recombination we have developed a new system to conditionally regulate the blm allele for introduction of biallelic mutations across the genome transient deficiency of blm induces homologous recombination not only between sister chromatids but also between homologous chromosomes resulting in a high rate of loss of heterozygosity loh introduction of genomewide mutations in escs can be achieved by retroviral vector in combination using genomewide mutagenesis and transient loss of blm expression we have generated es libraries with biallelic mutations these results show that this new system is very efficient for identifying gene functions in escs