Guanjun Gao, Natalia Wesolowska, and Yikang S. Rong from the laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, have come out with a novel technique to knock out gene of interest in Drosophila. Its called SIRT , which combines various existing methods like homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for targeted mutagenesis.

In SIRT, homologous recombination is used to place a landing site for the phage phiC31 integrase in the vicinity of the target locus. All subsequent genetic modifications to the same gene are introduced by integrase-mediated precise insertion of plasmids directly injected into embryos.

Targeted mutagenesis through homologous recombination has allowed researchers to mutate a particular locus in Drosophila in any desired way. SIRT expands this approach by targeting the attP landing site to the locus of interest.

The greatest obstacle that one faces when using the SIRT method is the construction of the appropriate vectors that contain complex arrangements of several DNA elements. We simplify the process by taking advantage of the versatile bacterial recombineering methodology. This method increases flexibility and bypasses the shortcomings of traditional cloning, because it does not rely on the availability of restriction cut sites at the site of modification.— Guanjun Gao, Natalia Wesolowska, and Yikang S. Rong

This protocol was recently published in Cold Spring Harbour Protocols and available freely to all readers. The entire protocol can be read here

Reference : Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5236

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