The efficiency of nuclear plasmid DNA delivery is a critical determinant of transgene expression at the single cell level

Dominic Glover, Denisse L Leyton, Gregory William Moseley, David Andrew Jans

Research output: Contribution to journalArticleResearchpeer-review

Abstract

BACKGROUND: The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression. METHODS: Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery. RESULTS: PEI was found to mediate a greater and more rapid nuclear accumulation of plasmid DNA compared to LFA, but reporter gene expression was shown to be higher for LFA than PEI when an equivalent number of plasmids were in the nucleus. Sorting of the extracted nuclei according to the level of reporter expression demonstrated that reporter expression was dependent upon the number of plasmids delivered into the nucleus, with both threshold and saturation in expression evident with few or many nuclear plasmids. CONCLUSIONS: Our findings demonstrate formally that although the efficiency of plasmid nuclear delivery is a critical determinant of the level of transgene expression, intranuclear events also influence the transcriptional activity of the transgene, and must be taken into consideration when attempting to maximize the efficiency of non-viral vectors.
Original languageEnglish
Pages (from-to)77 - 85
Number of pages8
JournalJournal of Gene Medicine
Volume12
DOIs
Publication statusPublished - 2010

Cite this

@article{20efdd934b6b48568098c5bc7ac2d007,
title = "The efficiency of nuclear plasmid DNA delivery is a critical determinant of transgene expression at the single cell level",
abstract = "BACKGROUND: The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression. METHODS: Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery. RESULTS: PEI was found to mediate a greater and more rapid nuclear accumulation of plasmid DNA compared to LFA, but reporter gene expression was shown to be higher for LFA than PEI when an equivalent number of plasmids were in the nucleus. Sorting of the extracted nuclei according to the level of reporter expression demonstrated that reporter expression was dependent upon the number of plasmids delivered into the nucleus, with both threshold and saturation in expression evident with few or many nuclear plasmids. CONCLUSIONS: Our findings demonstrate formally that although the efficiency of plasmid nuclear delivery is a critical determinant of the level of transgene expression, intranuclear events also influence the transcriptional activity of the transgene, and must be taken into consideration when attempting to maximize the efficiency of non-viral vectors.",
author = "Dominic Glover and Leyton, {Denisse L} and Moseley, {Gregory William} and Jans, {David Andrew}",
year = "2010",
doi = "10.1002/jgm.1406",
language = "English",
volume = "12",
pages = "77 -- 85",
journal = "Journal of Gene Medicine",
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publisher = "Wiley-Blackwell",

}

The efficiency of nuclear plasmid DNA delivery is a critical determinant of transgene expression at the single cell level. / Glover, Dominic; Leyton, Denisse L; Moseley, Gregory William; Jans, David Andrew.

In: Journal of Gene Medicine, Vol. 12, 2010, p. 77 - 85.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The efficiency of nuclear plasmid DNA delivery is a critical determinant of transgene expression at the single cell level

AU - Glover, Dominic

AU - Leyton, Denisse L

AU - Moseley, Gregory William

AU - Jans, David Andrew

PY - 2010

Y1 - 2010

N2 - BACKGROUND: The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression. METHODS: Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery. RESULTS: PEI was found to mediate a greater and more rapid nuclear accumulation of plasmid DNA compared to LFA, but reporter gene expression was shown to be higher for LFA than PEI when an equivalent number of plasmids were in the nucleus. Sorting of the extracted nuclei according to the level of reporter expression demonstrated that reporter expression was dependent upon the number of plasmids delivered into the nucleus, with both threshold and saturation in expression evident with few or many nuclear plasmids. CONCLUSIONS: Our findings demonstrate formally that although the efficiency of plasmid nuclear delivery is a critical determinant of the level of transgene expression, intranuclear events also influence the transcriptional activity of the transgene, and must be taken into consideration when attempting to maximize the efficiency of non-viral vectors.

AB - BACKGROUND: The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression. METHODS: Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery. RESULTS: PEI was found to mediate a greater and more rapid nuclear accumulation of plasmid DNA compared to LFA, but reporter gene expression was shown to be higher for LFA than PEI when an equivalent number of plasmids were in the nucleus. Sorting of the extracted nuclei according to the level of reporter expression demonstrated that reporter expression was dependent upon the number of plasmids delivered into the nucleus, with both threshold and saturation in expression evident with few or many nuclear plasmids. CONCLUSIONS: Our findings demonstrate formally that although the efficiency of plasmid nuclear delivery is a critical determinant of the level of transgene expression, intranuclear events also influence the transcriptional activity of the transgene, and must be taken into consideration when attempting to maximize the efficiency of non-viral vectors.

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