Active recombinant Tol2 transposase for gene transfer and gene discovery applications.

Jun Ni,David Nelsen,Mark D Urban,Stephen C Ekker,Darius Balciunas,Kimberly J Skuster,Kirk J Wangensteen

doi:10.1186/s13100-016-0062-z

Abstract

BackgroundThe revolutionary concept of “jumping genes” was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. Transposable elements (TEs) represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock’s Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. Other members of the hAT superfamily include the insect element Hermes and Tol2 from medaka. In recent years, genetic tools derived from the ‘cut’ and ‘paste’ Tol2 DNA transposon have been widely used for genomic manipulation in zebrafish, mammals and in cells in vitro.ResultsWe report the purification of a functional recombinant Tol2 protein from E.coli. We demonstrate here that following microinjection using a zebrafish embryo test system, purified Tol2 transposase protein readily catalyzes gene transfer in both somatic and germline tissues in vivo. We show that purified Tol2 transposase can promote both in vitro cutting and pasting in a defined system lacking other cellular factors. Notably, our analysis of Tol2 transposition in vitro reveals that the target site preference observed for Tol2 in complex host genomes is maintained using a simpler target plasmid test system, indicating that the primary sequence might encode intrinsic cues for transposon integration.ConclusionsThis active Tol2 protein is an important new tool for diverse applications including gene discovery and molecular medicine, as well as for the biochemical analysis of transposition and regulation of hAT transposon/genome interactions. The measurable but comparatively modest insertion site selection bias noted for Tol2 is largely determined by the primary sequence encoded in the target sequence as assessed through studying Tol2 protein-mediated transposition in a cell-free system.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0062-z) contains supplementary material, which is available to authorized users.

Highlights

The revolutionary concept of “jumping genes” was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize
We demonstrate that the highest activity variant can be epitope-tagged and retain full activity, and we purify epitope-tagged Tol2 protein (His-Tol2) from E. coli
When a copy of medaka Tol2 genomic sequence was introduced into zebrafish cells that do not harbor any endogenous Tol2 elements, a third Tol2 isoform of different length was identified (Tol2-M) [17] (Fig. 1a)

Summary

Introduction

The revolutionary concept of “jumping genes” was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. McClintock’s Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. The autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam from snapdragon define an ancient and diverse DNA transposon superfamily named hAT [2,3,4]. Previous research has suggested that the mechanisms of target site selection are very complex and varied from mobile element to mobile element. In many cases, it involves the direct interaction between the transposase/recombinase and the target DNA or their indirect communication through accessory proteins [11]. Specific factors that contribute to hAT element integration preference are largely unknown

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