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Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes

Abstract

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene–operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing 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 transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1 , and Ty1) have been described, which bypass many limitations of in vivo systems2,3,4,5,6,7. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.

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Figure 1: Transposome formation.
Figure 2: DNA sequence analysis of candidate inserts indicate that they represent bona fide Tn5-like transposition events.
Figure 3: Southern blot analysis of transposition clones in P. vulgaris. Hpa I/EcoRV digests of genomic DNAs isolated from presumed transposition clones were subjected to Southern blot analysis using as probes 23-mer and 25-mer oligonucleotides that correspond to the two ends of the transposon.

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Acknowledgements

We wish to thank Erich Moan and Joanne Decker for their technical assistance and input, Keith Derbyshire for communicating results prior to publication, and George Buriakov and Guy Plunkett for help with the E. coli insert analysis. Some of the reported experiments were supported by a grant (GM50692) from the National Institutes of Health to W.S.R. A preliminary report of the S. cerevisiae experiments can be found in Hoffman et al., Curr. Genet .35, 305 (1999).

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Correspondence to William S. Reznikoff.

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Goryshin, I., Jendrisak, J., Hoffman, L. et al. Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat Biotechnol 18, 97–100 (2000). https://doi.org/10.1038/72017

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