A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila. Export

@article{citeulike:3201283, abstract = {Gene targeting provides a powerful tool for dissecting gene function. However, repeated targeting of a single locus remains a practice mostly limited to unicellular organisms that afford simple targeting methodologies. We developed an efficient method to repeatedly target a single locus in Drosophila. In this method, which we term "site-specific integrase mediated repeated targeting" (SIRT), an attP attachment site for the phage phiC31 integrase is first targeted to the vicinity of the gene of interest by homologous recombination. All subsequent modifications of that gene are introduced by phiC31-mediated integration of plasmids carrying an attB attachment site and the desired mutation. This highly efficient integration results in a tandem duplication of the target locus, which is then reduced into a single copy carrying the mutation, likely by the efficient "single strand annealing" mechanism, induced with a DNA double-strand break (DSB). We used SIRT to generate a series of six mutations in the Drosophila nbs gene, ranging from single amino acid replacements and small in-frame deletions to complete deletion of the gene. Because all of the components of SIRT are functional in many different organisms, it is readily adaptable to other multicellular organisms.}, address = {Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.}, author = {Gao, Guanjun and McMahon, Conor and Chen, Jie and Rong, Yikang S S.}, citeulike-article-id = {3201283}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.0805843105}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/18772376}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=18772376}, day = {4}, doi = {10.1073/pnas.0805843105}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {attp, drosophila, dsb, mutagenesis, phic31, recombination, sirt, targeting}, month = {September}, posted-at = {2008-09-07 19:42:02}, priority = {2}, title = {A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila.}, url = {http://dx.doi.org/10.1073/pnas.0805843105}, year = {2008} }

TY - JOUR ID - citeulike:3201283 L3 - citeulike-article-id:3201283 N2 - Gene targeting provides a powerful tool for dissecting gene function. However, repeated targeting of a single locus remains a practice mostly limited to unicellular organisms that afford simple targeting methodologies. We developed an efficient method to repeatedly target a single locus in Drosophila. In this method, which we term "site-specific integrase mediated repeated targeting" (SIRT), an attP attachment site for the phage phiC31 integrase is first targeted to the vicinity of the gene of interest by homologous recombination. All subsequent modifications of that gene are introduced by phiC31-mediated integration of plasmids carrying an attB attachment site and the desired mutation. This highly efficient integration results in a tandem duplication of the target locus, which is then reduced into a single copy carrying the mutation, likely by the efficient "single strand annealing" mechanism, induced with a DNA double-strand break (DSB). We used SIRT to generate a series of six mutations in the Drosophila nbs gene, ranging from single amino acid replacements and small in-frame deletions to complete deletion of the gene. Because all of the components of SIRT are functional in many different organisms, it is readily adaptable to other multicellular organisms. JF - Proceedings of the National Academy of Sciences of the United States of America SN - 1091-6490 AD - Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. TI - A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila. KW - attp KW - drosophila KW - dsb KW - mutagenesis KW - phic31 KW - recombination KW - sirt KW - targeting AU - Gao, Guanjun AU - McMahon, Conor AU - Chen, Jie AU - Rong, Yikang S PY - 2008/09/04/ UR - http://dx.doi.org/10.1073/pnas.0805843105 ER -