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

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 DROSOPHIL...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2008-09, Vol.105 (37), p.13999-14004
Main Authors: Gao, Guanjun, McMahon, Conor, Chen, Jie, Rong, Yikang S
Format: Article
Language:English
Subjects:
Alleles
Amino acids
Animals
Biological Sciences
DNA damage
Drosophila
Drosophila melanogaster - genetics
Efficiency loss
Female
Gene Deletion
Gene loci
Gene targeting
Genetic loci
Genetic mutation
Genetic vectors
Genomics
homologous recombination
Insects
Integrase
Integration
Male
Mutagenesis
Mutagenesis - genetics
Mutation
Phages
Plasmids
Polymerase chain reaction
Recombination, Genetic - genetics
targeted mutagenesis
Transgenes
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0805843105