Manipulating piggyBac Transposon Chromosomal Integration Site Selection in Human Cells
| Autor: | Matthew H. Wilson, Alfred L. George, Claudia Kettlun, Aparna Kaja, Daniel L. Galvan |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Chromatin Immunoprecipitation
animal structures Blotting Western Genetic Vectors Transposases Computational biology Gene delivery Biology Real-Time Polymerase Chain Reaction Transfection Genome 03 medical and health sciences 0302 clinical medicine Plasmid Drug Discovery Genetics Chromosomes Human Humans Molecular Biology Gene Transposase 030304 developmental biology Pharmacology 0303 health sciences Genome Human Transposon integration fungi Zinc Fingers DNA-Binding Proteins HEK293 Cells PiggyBac Transposon System 030220 oncology & carcinogenesis DNA Transposable Elements Mutagenesis Site-Directed Molecular Medicine Original Article Human genome Genetic Engineering Plasmids |
| Zdroj: | Molecular Therapy. 19:1636-1644 |
| ISSN: | 1525-0016 |
| DOI: | 10.1038/mt.2011.129 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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