title: An optimized, broadly applicable piggyBac transposon induction system.
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.
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Zongtai
authors.initials: Z
authors.lastname: Qi
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Michael Nathaniel
authors.initials: MN
authors.lastname: Wilkinson
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Xuhua
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Sumithra
authors.initials: S
authors.lastname: Sankararaman
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA.
authors.forename: David
authors.initials: D
authors.lastname: Mayhew
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Robi David
authors.initials: RD
authors.lastname: Mitra
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D004251:DNA Transposable Elements; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000235:genetics; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic; D019895:Transposases / Q000235:genetics* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D009025:Morpholines; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; C540198:Shield-1 compound; D013629:Tamoxifen; C016601:afimoxifene; D013762:Tetrahydrofolate Dehydrogenase; D019895:Transposases; D022021:Tacrolimus Binding Proteins