title: A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBac transposase.
abstract: The piggyBac (PB) transposable element has recently accumulated enormous attention as a tool for the transgenesis in various eukaryotic organisms. Arginine-rich cell-penetrating peptides (CPPs) are protein transduction domains containing a large amount of basic amino acids that were found to be capable of delivering biologically active macromolecules into living cells. In this study, we demonstrate a strategy, which we called "transposoduction", which is a one-plasmid gene delivery system mediated by the nontoxic CPP-piggyBac transposase (CPP-PBase) fusion protein to accomplish both protein transduction and transposition. CPPs were proven to be able to synchronously deliver covalently linked PBase and noncovalently linked a cis plasmid into human cells. The expression of promoterless reporter genes coding for red (dTomato) and yellow (mOrange) fluorescent proteins (RFP and YFP) with PB elements could be detected in cells treated with the PBase-expressing plasmid after 3 days indicating transposition of coding regions to downstream of endogenous promoter sequences. An enhanced green fluorescent protein (EGFP) plasmid-based excision assay further confirmed the efficiency of the bifunctional CPP-PBase fusion protein. In conclusion, this strategy representing a combinational concept of both protein transduction and mobile transposition may provide tremendous potential for safe and efficient cell line transformation, gene therapy and functional genomics.
authors.affiliation: Department of Natural Resources and Environmental Studies, National Dong Hwa University, No. 1, Sec. 2, Da-Hsueh Road, Shoufeng, Hualien 97401, Taiwan.
authors.forename: Cheng-Yi
authors.initials: CY
authors.lastname: Lee
authors.forename: Jheng-Fong
authors.initials: JF
authors.lastname: Li
authors.forename: Ji-Sing
authors.initials: JS
authors.lastname: Liou
authors.forename: Yuh-Chyang
authors.initials: YC
authors.lastname: Charng
authors.forename: Yue-Wern
authors.initials: YW
authors.lastname: Huang
authors.forename: Han-Jung
authors.initials: HJ
authors.lastname: Lee
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D014162:Transfection; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D057846:Cell-Penetrating Peptides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019895:Transposases