Combining “Donor-” and “Helper-” Plasmids for piggyBac Transformation.

Dr. Alfred Handler

Alfred Handler, Ph.D., Research Geneticist (Insects), Agricultural Research Service, U.S. Department of Agriculture, Gainesville, Florida More about the author.

The piggyBac transposon was first tested as a germline transformation system in insects, but has also been shown to be a powerful tool in the transformation of other organisms, and in particular, its use in reprogramming mammalian somatic cells into pluripotent stem cells.

The typical binary vector/helper plasmid transformation system routinely used in insects, however, may be less than ideal for mammalian systems where DNA delivery is less efficient, requiring modifications for routine gene-transfer in particular host organisms.

A typical DNA-transposon and a typical binary gene vector system derived from a DNA-tranposon.

Recently, Chakraborty et al. (2014) reported the development and testing of a new efficient single-plasmid transformation system for mammalian cell lines.

Combining vector and transposase helper sequences in a single plasmid is possible in various configurations, but vector stability requires that the transposase is either deleted or inactivated during, or soon after transposition. In this report, inactivation is achieved by having the transposase and the vector marker gene share the same polyA termination sequence, such that transposition of the vector inactivates the external transposase helper. To achieve this, a hyperactive piggyBac transposase (hyPBase) coding region, regulated by a CMV promoter but lacking a polyA termination sequence, was inserted upstream to the vector 5’ and 3’ inverted terminal repeat regions having a ubiquitin-regulated GFP-polyA gene inserted within the transposon ends. The transposition frequency of this construct was favorable compared to similar constructs where the transposase gene had its own polyA site. To eliminate any cells having residual transposase expression, bi-cistronic expression of HSV-tk along with transposase allowed any co-expressing cells to be killed by ganciclovir.

One-plasmid vs Two-plasmid piggyBac system.  see doi:10.1038/srep07403

One-plasmid vs Two-plasmid piggyBac system. see doi:10.1038/srep07403

These modifications will be most useful for transformation of non-insect systems, and especially cell lines, though for specific cases in insects and other organisms where a single vector/helper plasmid is most efficacious, the transposase inactivation system may prove highly useful. It also provides another means of controlling vector plasmid-encoded gene expression pre- and post-integration.

 Chakraborty S, Ji H, Chen J, Gersbach CA, Leong KW (2014) Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis. Sci Rep 4  10.1038/srep07403

More about the author.

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