Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

DNA transposition is an important biological phenomenon that mediates genom e rearrangements, inheritance of antibiotic resistance determinants, and in tegration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mut ations, generating gene-operon fusions to reporter functions, providing phy sical or genetic landmarks for the cloning of adjacent DNAs, and locating p rimer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the trans poson of choice and the gene encoding the transposase responsible for catal yzing the transposition have to be introduced into the cell to be studied ( microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the tra nsposase must be expressed in the target host, the transposon must be intro duced into the host on a suicide vector, and the transposase usually is exp ressed in subsequent generations, resulting in potential genetic instabilit y. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, an d Ty1) have been described, which bypass many limitations of in vivo system s(2-6). For this purpose, we have developed a technique for transposition t hat involves the formation in vitro of released Tn5 transposition complexes (Transposomes(TM)) followed by introduction of the complexes into the targ et cell of choice by electroporation. In this report, we show that this sim ple, robust technology can generate high-efficiency transposition in all te sted bacterial species (Escherichia coli, Salmonella typhimurium, and Prote us vulgaris) We also isolated transposition events in the yeast Saccharomyc es cerevisiae.