piggyBac’s Genomic Effects

The transposon piggyBac is a very popular platform for moving transgenes into the genomes of a wide range of organisms.  In an upcoming issue of Nucleic Acids Research Saha et al. address a number of important questions regarding the potential for the piggyBac system to have undesirable effects on the host genome – in this case, the human genome.


Typical DNA transposon and its conversion to a binary gene vector system

Those working with or considering working with piggyBac in insects probably will have similar questions and this work will be of some interest.

The human genome has some 2000 piggyBac-like elements and this raises the question of whether introducing the piggyBac system could result in mobilization of endogenous elements or genome rearrangements.  More generally, can piggyBac transposase increase the frequency of double-stranded DNA breaks.  Saha et al. also looked at the frequency with which the plasmid backbone in which piggyBac is cloned becomes integrated.  Finally, they ask whether the terminal repeats of piggyBac have any promoter or enhancer activity.

While these question are specifically addressed in human cells, they are clearly of interest generally.  For the most part, the answers to these questions are all essentially negative.

Mechanism of piggyBac transposition.   Image credit Skipper et al. Journal of Biomedical Science 2013 20:92   doi:10.1186/1423-0127-20-92

Mechanism of piggyBac transposition. Image credit Skipper et al. Journal of Biomedical Science 2013 20:92 doi:10.1186/1423-0127-20-92

No increase in the number of double-stranded DNA breaks could be detected in the presence of piggyBac transposase, nor was there any evidence of the 2000 piggyBac-like elements interacting with piggyBac transposase.

piggyBac vectors and piggyBac transposase can be supplied using two plasmids or a single plasmid.  The single-plasmid  ‘cis’ configuration of the vector and transposase has been shown, at least in human cells, to be more effective.  See a recent IGTRCN Technology Topics post by Al Handler.  But the plasmid backbone can integrate following piggyBac excision and if the backbone contains the coding region of piggyBac transposase then a continuous source of transposase will be present resulting in destabilizing the integrated vector.  So, there is a clear trade-off in using ‘cis’ versus ‘trans’ configurations of the vector and transposase.  This questions have not been addressed in insect cells.

Saha et al. present data that indicate that the inverted repeats of piggyBac have no enhancer activity.  However, they did find that the 5′ inverted repeat  (just the 17bp sequence) had very weak promoter activity directed toward the ‘inside’ of the vector.

Finally, the authors show that they can remove the cryptic splice-site (at least in human cells) that is present in the 5′ terminal repeat without affecting the functioning of the terminal sequences.

Overall, the absence of any ‘cross reactivity’ between the piggyBac system and endogenous piggyBac-like elements in the human genome is good news for those considering the use of this system as a human therapeutic.

For insect biologists, this work raises important questions that they too should at least consider when planning to use a transposon-based system like piggyBac.  Most importantly, are there endogenous piggyBac or piggyBac-like elements in ‘your’ genome?  With the growing abundance of genome sequence data addressing this question becomes routine.

Sunandan Saha, Lauren E. Woodard, Elizabeth M. Charron, Richard C. Welch, Cliona M. Rooney, and Matthew H. Wilson, Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells Nucl. Acids Res. first published online January 20, 2015 doi:10.1093/nar/gkv017 


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