title: Manipulating piggyBac transposon chromosomal integration site selection in human cells.
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.
authors: Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.
authors: Claudia
authors: C
authors: Kettlun
authors: Daniel L
authors: DL
authors: Galvan
authors: Alfred L
authors: AL
authors: George
authors: Aparna
authors: A
authors: Kaja
authors: Matthew H
authors: MH
authors: Wilson
mesh_terms: D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation / Q000379:methods; D002877:Chromosomes, Human / Q000235:genetics*; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D019895:Transposases