DNA nuclear targeting sequences for non-viral gene delivery

Ethlinn V B van Gaal; Ronald S Oosting; Roel van Eijk; Marta Bakowska; Dries Feyen; Robbert Jan Kok; Wim E Hennink; Daan J A Crommelin; Enrico Mastrobattista

doi:10.1007/s11095-011-0407-8

DNA nuclear targeting sequences for non-viral gene delivery

Ethlinn V B van Gaal, Ronald S Oosting, Roel van Eijk, Marta Bakowska, Dries Feyen, Robbert Jan Kok, Wim E Hennink, Daan J A Crommelin, Enrico Mastrobattista

Research output: Contribution to journal › Article › Academic › peer-review

49 Citations (Scopus)

Abstract

PURPOSE: To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.

METHODS: A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.

RESULTS: Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.

CONCLUSION: No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.

Original language	English
Pages (from-to)	1707-22
Number of pages	16
Journal	Pharmaceutical Research
Volume	28
Issue number	7
DOIs	https://doi.org/10.1007/s11095-011-0407-8
Publication status	Published - Jul-2011
Externally published	Yes

Keywords

Base Sequence
Cell Line, Tumor
Cell Nucleus
Electroporation
Flow Cytometry
Gene Transfer Techniques
Genetic Vectors
HeLa Cells
Humans
Molecular Sequence Data
Plasmids
Polymerase Chain Reaction

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@article{a02cbd5e77b541fbb57d30449682c281,

title = "DNA nuclear targeting sequences for non-viral gene delivery",

abstract = "PURPOSE: To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.METHODS: A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.RESULTS: Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.CONCLUSION: No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.",

keywords = "Base Sequence, Cell Line, Tumor, Cell Nucleus, Electroporation, Flow Cytometry, Gene Transfer Techniques, Genetic Vectors, HeLa Cells, Humans, Molecular Sequence Data, Plasmids, Polymerase Chain Reaction",

author = "{van Gaal}, {Ethlinn V B} and Oosting, {Ronald S} and {van Eijk}, Roel and Marta Bakowska and Dries Feyen and Kok, {Robbert Jan} and Hennink, {Wim E} and Crommelin, {Daan J A} and Enrico Mastrobattista",

year = "2011",

month = jul,

doi = "10.1007/s11095-011-0407-8",

language = "English",

volume = "28",

pages = "1707--22",

journal = "Pharmaceutical Research",

issn = "0724-8741",

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TY - JOUR

T1 - DNA nuclear targeting sequences for non-viral gene delivery

AU - van Gaal, Ethlinn V B

AU - Oosting, Ronald S

AU - van Eijk, Roel

AU - Bakowska, Marta

AU - Feyen, Dries

AU - Kok, Robbert Jan

AU - Hennink, Wim E

AU - Crommelin, Daan J A

AU - Mastrobattista, Enrico

PY - 2011/7

Y1 - 2011/7

N2 - PURPOSE: To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.METHODS: A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.RESULTS: Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.CONCLUSION: No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.

AB - PURPOSE: To evaluate if introduction of DNA nuclear Targeting Sequences (DTS; i.e. recognition sequences for endogenous DNA-binding proteins) in plasmid DNA (pDNA) leads to increased transfection efficiency of non-viral gene delivery by virtue of enhanced nuclear import of the pDNA.METHODS: A set of DTS was identified and cloned into EGFP-reporter plasmids controlled by the CMV-promoter. These pDNA constructs were delivered into A431 and HeLa cells using standard electroporation, pEI-based polyfection or lipofection methods. The amount of pDNA delivered into the nucleus was determined by qPCR; transfection efficiency was determined by flow cytometry.RESULTS: Neither of these DTS increased transgene expression. We varied several parameters (mitotic activity, applied dose and delivery strategy), but without effect. Although upregulated transgene expression was observed after stimulation with TNF-α, this effect could be ascribed to non-specific upregulation of transcription rather than enhanced nuclear import. Nuclear copy numbers of plasmids containing or lacking a DTS did not differ significantly after lipofectamine-based transfection in dividing and non-dividing cells.CONCLUSION: No beneficial effects of DTS on gene expression or nuclear uptake were observed in this study.

KW - Base Sequence

KW - Cell Line, Tumor

KW - Cell Nucleus

KW - Electroporation

KW - Flow Cytometry

KW - Gene Transfer Techniques

KW - Genetic Vectors

KW - HeLa Cells

KW - Humans

KW - Molecular Sequence Data

KW - Plasmids

KW - Polymerase Chain Reaction

U2 - 10.1007/s11095-011-0407-8

DO - 10.1007/s11095-011-0407-8

M3 - Article

C2 - 21424159

SN - 0724-8741

VL - 28

SP - 1707

EP - 1722

JO - Pharmaceutical Research

JF - Pharmaceutical Research

IS - 7

ER -

DNA nuclear targeting sequences for non-viral gene delivery

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