A highly efficient recombineering-based method for generating conditional knockout (cko) mutations in mice is described. This method utilizes homologous recombination mediated by λ phage Red proteins to subclone DNA from BACs into high-copy plasmids via gap repair. It also incorporates Cre or Flp recombinases to introduce loxP or FRT sites into the subcloned DNA. The method uses longer homology regions compared to previous techniques, enhancing efficiency and reliability. New E. coli strains with temperature-sensitive λ prophage and arabinose-inducible Cre or Flp recombinases are used. Two new Neo selection cassettes are also introduced, effective in both E. coli and mouse ES cells. This method allows for the rapid generation of cko-targeting vectors in less than two weeks, facilitating the creation of knock-in mutations and transgene constructs. The method also aids in analyzing regulatory elements and functional domains of genes. Conditional knockout mice allow for gene inactivation in a tissue- or temporal-specific manner, overcoming the issue of embryonic lethality caused by complete gene deficiency. The method involves subcloning BAC DNA into plasmids, introducing loxP sites, and using Cre recombinase to excise the gene. The process includes generating targeting vectors, introducing loxP sites, and excising the Neo cassette. The method is efficient, reliable, and enables the generation of cko-targeting vectors with high targeting efficiency. The method is applicable for generating cko-targeting vectors for various genes, including Evi9, and has been successfully used to create conditional knockout alleles. The method is based on recombineering, which allows for the modification of DNA without restriction enzymes or DNA ligases, making it a powerful tool for functional genomic studies. The method involves PCR amplification of homology arms, subcloning into plasmids, and using recombinases to introduce target sites. The process is efficient and allows for the rapid generation of cko-targeting vectors, facilitating the study of gene function in the post-genome era.A highly efficient recombineering-based method for generating conditional knockout (cko) mutations in mice is described. This method utilizes homologous recombination mediated by λ phage Red proteins to subclone DNA from BACs into high-copy plasmids via gap repair. It also incorporates Cre or Flp recombinases to introduce loxP or FRT sites into the subcloned DNA. The method uses longer homology regions compared to previous techniques, enhancing efficiency and reliability. New E. coli strains with temperature-sensitive λ prophage and arabinose-inducible Cre or Flp recombinases are used. Two new Neo selection cassettes are also introduced, effective in both E. coli and mouse ES cells. This method allows for the rapid generation of cko-targeting vectors in less than two weeks, facilitating the creation of knock-in mutations and transgene constructs. The method also aids in analyzing regulatory elements and functional domains of genes. Conditional knockout mice allow for gene inactivation in a tissue- or temporal-specific manner, overcoming the issue of embryonic lethality caused by complete gene deficiency. The method involves subcloning BAC DNA into plasmids, introducing loxP sites, and using Cre recombinase to excise the gene. The process includes generating targeting vectors, introducing loxP sites, and excising the Neo cassette. The method is efficient, reliable, and enables the generation of cko-targeting vectors with high targeting efficiency. The method is applicable for generating cko-targeting vectors for various genes, including Evi9, and has been successfully used to create conditional knockout alleles. The method is based on recombineering, which allows for the modification of DNA without restriction enzymes or DNA ligases, making it a powerful tool for functional genomic studies. The method involves PCR amplification of homology arms, subcloning into plasmids, and using recombinases to introduce target sites. The process is efficient and allows for the rapid generation of cko-targeting vectors, facilitating the study of gene function in the post-genome era.