Manipulating piggyBac transposon chromosomal integration site selection in human cells

Mol Ther. 2011 Sep;19(9):1636-44. doi: 10.1038/mt.2011.129. Epub 2011 Jul 5.

Abstract

The ability to direct gene delivery to a user-defined chromosomal location would greatly improve gene transfer applications. The piggyBac transposon system is a nonviral gene transfer system proven effective in a variety of cells and tissues, including human cells. We fused a highly site-specific synthetic zinc-finger DNA-binding domain (ZFP) to the N-terminus of the piggyBac transposase and evaluated site-directed genomic integration in human cells. Chimeric ZFP-piggyBac transposase exhibited robust gene transfer activity, targeted binding to a cognate endogenous chromosomal ZFP site in the human genome, and site-directed transposon integration into an episomal plasmid target containing a single ZFP site in human cells. We evaluated the ability of ZFP-piggyBac to direct gene integration into an engineered chromosomal ZFP target site in the human genome and consistently observed a higher degree of ZFP-piggyBac site-directed genomic integration when compared to native piggyBac. Chromatin immunoprecipitation (ChIP) experiments revealed binding of native piggyBac to our engineered TTAA-containing chromosomal target which supported integration, but not a TTAA-deficient chromosomal target which lacked integration. Our results offer insight into the requirements for using a chimeric zinc finger-piggyBac transposase to direct integration into a user-defined chromosomal location.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Chromatin Immunoprecipitation / methods
  • Chromosomes, Human / genetics*
  • DNA Transposable Elements*
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Genetic Engineering / methods*
  • Genetic Vectors
  • Genome, Human
  • HEK293 Cells
  • Humans
  • Mutagenesis, Site-Directed
  • Plasmids
  • Real-Time Polymerase Chain Reaction
  • Transfection
  • Transposases / genetics
  • Transposases / metabolism
  • Zinc Fingers

Substances

  • DNA Transposable Elements
  • DNA-Binding Proteins
  • Transposases