Manipulating piggybac transposon chromosomal integration site selection in human cells

Claudia Kettlun, Daniel L. Galvan, Alfred L. George Jr, Aparna Kaja, Matthew H. Wilson*

*Corresponding author for this work

Research output: Contribution to journalArticle

47 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1636-1644
Number of pages9
JournalMolecular Therapy
Volume19
Issue number9
DOIs
StatePublished - Sep 2011

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Pharmacology
  • Drug Discovery

Fingerprint Dive into the research topics of 'Manipulating piggybac transposon chromosomal integration site selection in human cells'. Together they form a unique fingerprint.

  • Cite this