Protocol

SIRT Combines Homologous Recombination, Site-Specific Integration, and Bacterial Recombineering for Targeted Mutagenesis in Drosophila

Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA

¹ These authors contributed equally to this work.

²Corresponding author (rongy{at}mail.nih.gov)

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.