standard Active Recombinant Tol2 Transposase

Ni et al (2016) report successfully expressing and purifying Tol2 transposase and demonstrate its activity in vitro and in vivo.  Although they were working with zebrafish the system they describe might be of some interest to insect biologists.

medaka

Medaka, Oryzias latipes

Tol2 is a transposon in the hAT superfamily, along with founding members hobo, Ac and Tam3.  This is a large group of transposons that includes Hermes, a useful insect gene vector.  All members of this superfamily that have been tested have shown a broad host range extending well beyond the taxonomic group from which the element was first isolated.

Tol2 was originally isolated from the medaka fish (Oryzias latipes) and has been a useful gene vector in zebrafish fish and frog, as well as chicken, mouse and human cell cultures.

Tol2 showing exon/intron structure and inverted repeats.

Tol2 showing exon/intron structure.

Transposons are typically deployed as vectors as part of a binary system comprised of a non-autonomous transposon serving as the vector (nonautonomous refers to the fact that it can not produce its own transposase and depends on it to be supplied in trans) and a source of transposase.  Transposase is provided by either transiently expressing the transposase gene from a plasmid co-introduced into the target cells or by injecting transposase mRNA.

Tn5 transposition showing transposase binding to termini, synaptic complex formation, cleavage and target capture.

Tn5 transposition showing transposase binding to termini, synaptic complex formation, cleavage and target capture.

Having available purified active transposase is attractive because it might improve frequencies of integration perhaps by allowing integrations to occur immediately upon delivery to cells as opposed to the obligatory delay associated with transiently expressing transposase transgenes.

Transposase proteins have been difficult to express and reconstitute and the work of Ni et al (2016) is interesting from that perspective.

While the Tol2 transposase purified and tested was active, side by side comparisons with transposase mRNA did not show any improvement in transposition frequencies.  In fact, they were somewhat lower.  While a bit disappointing, the authors suggest that their system has not been optimized and that varying the ratio of protein to vector-containing plasmid might improve the observed frequencies.

Another option not discussed by the authors is to mix the transposase with Tol2 vector DNA that resembles the excised form of the element instead of having the vector in a plasmid.  This would be similar to how commercially available Tn5 and Tn5 transposase has been used.  One might be injecting functional synaptic complexes and those might prove effective at increase Tol2 integration frequencies.

Ni, J. Wangensteen, K. J., Nelsen, D., Balciunas, D., Skuster, K., Urban, M. D, Ekker, S. C. (2016) Active recombinant Tol2 transposase for gene transfer and gene discovery applications. Mobile DNA 7:6 doi 10.1186/d13100-016-0062-z

 

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