Manipulating piggyBac transposon chromosomal integration site selection in human cells.

Kettlun C, Galvan DL, George AL, Kaja A, Wilson MH
Mol Ther. 2011 19 (9): 1636-44

PMID: 21730970 · PMCID: PMC3182353 · DOI:10.1038/mt.2011.129

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.

MeSH Terms (16)

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

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