Format

Send to

Choose Destination
See comment in PubMed Commons below
Nucleic Acids Res. 2017 Jan 12. pii: gkw1290. doi: 10.1093/nar/gkw1290. [Epub ahead of print]

An optimized, broadly applicable piggyBac transposon induction system.

Author information

  • 1Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA.
  • 2Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA rmitra@genetics.wustl.edu.

Abstract

The piggyBac (PB) transposon has been used in a number of biological applications. The insertion of PB transposons into the genome can disrupt genes or regulatory regions, impacting cellular function, so for many experiments it is important that PB transposition is tightly controlled. Here, we systematically characterize three methods for the post-translational control of the PB transposon in four cell lines. We investigated fusions of the PB transposase with ERT2 and two degradation domains (FKBP-DD, DHFR-DD), in multiple orientations, and determined (i) the fold-induction achieved, (ii) the absolute transposition efficiency of the activated construct and (iii) the effects of two inducer molecules on cellular transcription and function. We found that the FKBP-DD confers the PB transposase with a higher transposition activity and better dynamic range than can be achieved with the other systems. In addition, we found that the FKBP-DD regulates transposon activity in a reversible and dose-dependent manner. Finally, we showed that Shld1, the chemical inducer of FKBP-DD, does not interfere with stem cell differentiation, whereas tamoxifen has significant effects. We believe the FKBP-based PB transposon induction will be useful for transposon-mediated genome engineering, insertional mutagenesis and the genome-wide mapping of transcription factor binding.

PMID:
28082389
DOI:
10.1093/nar/gkw1290
[PubMed - as supplied by publisher]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Silverchair Information Systems
    Loading ...
    Support Center