Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection
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....
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| Published in | The journal of gene medicine Vol. 17; no. 1-2; pp. 33 - 53 |
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| Main Authors | , , |
| Format | Journal Article |
| Language | English |
| Published |
England
Blackwell Publishing Ltd
01.01.2015
Wiley Periodicals Inc |
| Subjects | |
| Online Access | Get full text |
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| Summary: | 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 (2 h of exposure) or between flow‐separated transfected cells (GFP+) and treated, untransfected cells (GFP–) at 8, 16 and 24 h 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 2 h; RAP1A and ACTA1 after 8 h; RAP1A, WDR78 and ACTA1 after 16 h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24 h. 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. Copyright © 2015 John Wiley & Sons, Ltd. |
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| Bibliography: | Phase images taken on Leica DMI 3000B (Bannockburn, IL, USA) to assess cell viability and morphology. HEK 293 T cells were seeded, allowed to adhere (approximately 18 h); then pharmacologic agent or vehicle control+ was delivered to the media above the cells and after a 1-h incubation, lipoplexes were formed and delivered to the cells. After 24 h, images were taken at × 100 magnification (scale bar is the same for all images and as shown is 200 µ M), with representative images shown. (A) Immunoglobin G 0.1 µ M, (B) eicosapentaenoic acid 3 µ M, (C) 5-episisomicin 0.1 µ M, (D) cytoclasin B 1 µ M, (E) apramycin 30 µ M, (F) tolazoline 0.1 µ M, (G) 4-hydroxytamoxifen 10 µ M, (H) benzoic acid 1 µ M, (I) folic acid 1 µ M, (J) hydrocortisone 10 µ M, (K) tetrachlorethylene 1 n M, (L) propanil 1 µ M, (M) 0.1 M NaCl vehicle control+, (N) MeOH vehicle control+, (O) ddH2O vehicle control+, (P) DMSO vehicle control+, (Q) 1 M NaOH vehicle control+ and (R) control-.Genes differentially expressed with a two-fold cut-off limit.Overlap of genes differentially expressed with a two-fold cut-off limit for polyplexes over time.Overlap of genes differentially expressed with a two-fold cut-off limit comparing polyplex gene expression profile with the lipoplex gene expression profile at each time point. ark:/67375/WNG-694LDS3R-5 National Science Foundation - No. CBET-1254415 Center for Nanohybrid Functional Materials - No. NSF EPS-1004094 ArticleID:JGM2822 American Heart Association - No. #10SDG2640217 USDA CSREES-Nebraska - No. NEB-21-146; No. NEB-26-211 istex:CCE32B357051BBF62B64B5911C4FF86635D6AA82 University of Nebraska Foundation (LaymanFunds) Nebraska Research Initiative, University of Nebraska-Lincoln Tobaccos Settlement Funds ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 1099-498X 1521-2254 |
| DOI: | 10.1002/jgm.2822 |