DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors.

Abstract:

:DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Cai Y,Bak RO,Krogh LB,Staunstrup NH,Moldt B,Corydon TJ,Schrøder LD,Mikkelsen JG

doi

10.1093/nar/gkt1163

subject

Has Abstract

pub_date

2014-02-01 00:00:00

pages

e28

issue

4

eissn

0305-1048

issn

1362-4962

pii

gkt1163

journal_volume

42

pub_type

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