Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection

Timothy M. Martin, Sarah A. Plautz, Angela K. Pannier

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Background: Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection. Methods: Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid-encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors. Results: Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2h; RAP1A and ACTA1 after 8h; RAP1A, WDR78 and ACTA1 after 16h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells. Conclusions: The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered.

Original languageEnglish (US)
Pages (from-to)33-53
Number of pages21
JournalJournal of Gene Medicine
Volume17
Issue number1-2
DOIs
StatePublished - Jan 1 2015

Fingerprint

Transcriptome
Transfection
Polymers
Lipids
Genes
Green Fluorescent Proteins
Polyethyleneimine
Gene Transfer Techniques
HEK293 Cells
Transgenes
Plasmids
DNA

Keywords

  • GFP
  • HEK 293
  • LF2000
  • Microarray analysis
  • Nonviral gene delivery
  • PEI
  • Temporal gene expression profile

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

Cite this

Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection. / Martin, Timothy M.; Plautz, Sarah A.; Pannier, Angela K.

In: Journal of Gene Medicine, Vol. 17, No. 1-2, 01.01.2015, p. 33-53.

Research output: Contribution to journalArticle

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abstract = "Background: Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection. Methods: Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid-encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors. Results: Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2h; RAP1A and ACTA1 after 8h; RAP1A, WDR78 and ACTA1 after 16h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells. Conclusions: The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered.",
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N2 - Background: Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection. Methods: Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid-encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors. Results: Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2h; RAP1A and ACTA1 after 8h; RAP1A, WDR78 and ACTA1 after 16h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells. Conclusions: The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered.

AB - Background: Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection. Methods: Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid-encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP-) at 8, 16 and 24h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors. Results: Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2h; RAP1A and ACTA1 after 8h; RAP1A, WDR78 and ACTA1 after 16h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells. Conclusions: The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered.

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