SIRT Combines Homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila
Open Access
- 1 June 2009
- journal article
- Published by Cold Spring Harbor Laboratory in Cold Spring Harbor Protocols
- Vol. 2009 (6)
- https://doi.org/10.1101/pdb.prot5236
Abstract
INTRODUCTION: Systematic mutational analysis is required for the comprehensive deciphering of gene function. However, repeated targeting of a single locus is labor intensive and has not been a routine approach for studies using multicellular organisms. We have developed the “site-specific integrase mediated repeated targeting” (SIRT) method to facilitate targeted mutagenesis in Drosophila melanogaster. 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. For SIRT mutagenesis, one must generate a series of plasmid vectors that contain various DNA elements placed at different positions in the target-homologous clone. Unlike traditional cloning methods, SIRT is not limited by the availability of convenient restriction cut sites. This protocol presents the details of SIRT plasmid construction, relying heavily on the method of bacterial recombineering and using a number of streamlined DNA elements.Keywords
This publication has 12 references indexed in Scilit:
- A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in DrosophilaProceedings of the National Academy of Sciences of the United States of America, 2008
- Methods for Homologous Recombination in DrosophilaPublished by Springer Science and Business Media LLC ,2008
- The Use of P-Element Transposons to Generate Transgenic FliesMethods in Molecular Biology, 2008
- An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrasesProceedings of the National Academy of Sciences of the United States of America, 2007
- Recombineering: In Vivo Genetic Engineering in E. coli, S. enterica, and BeyondMethods in enzymology, 2007
- P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogasterScience, 2006
- Site-Specific Transformation of Drosophila via ϕC31 Integrase-Mediated Cassette ExchangeGenetics, 2006
- Construction of Transgenic Drosophila by Using the Site-Specific Integrase From Phage C31Genetics, 2004
- Targeted mutagenesis by homologous recombination inD. melanogasterGenes & Development, 2002
- Gene Targeting by Homologous Recombination in DrosophilaScience, 2000