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

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DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors. / Cai, Yujia; Bak, Rasmus O.; Krogh, Louise Bechmann; Staunstrup, Nicklas H; Moldt, Brian; Corydon, Thomas J; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm.

In: Nucleic Acids Research, 21.11.2013.

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@article{8a469a0356fc4079b3e3f9d09c5d5ecb,
title = "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.",
author = "Yujia Cai and Bak, {Rasmus O.} and Krogh, {Louise Bechmann} and Staunstrup, {Nicklas H} and Brian Moldt and Corydon, {Thomas J} and Schr{\o}der, {Lisbeth Dahl} and Mikkelsen, {Jacob Giehm}",
year = "2013",
month = nov,
day = "21",
doi = "10.1093/nar/gkt1163",
language = "English",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",

}

RIS

TY - JOUR

T1 - DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors

AU - Cai, Yujia

AU - Bak, Rasmus O.

AU - Krogh, Louise Bechmann

AU - Staunstrup, Nicklas H

AU - Moldt, Brian

AU - Corydon, Thomas J

AU - Schrøder, Lisbeth Dahl

AU - Mikkelsen, Jacob Giehm

PY - 2013/11/21

Y1 - 2013/11/21

N2 - 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.

AB - 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.

U2 - 10.1093/nar/gkt1163

DO - 10.1093/nar/gkt1163

M3 - Journal article

C2 - 24270790

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

ER -