title: Temporal endogenous gene expression profiles in response to lipid-mediated transfection.
abstract: BACKGROUND
  Design of efficient nonviral gene delivery systems is limited as a result of the rudimentary understanding of the specific molecules and processes that facilitate DNA transfer.
  
  
  METHODS
  Lipoplexes formed with Lipofectamine 2000 (LF2000) and plasmid-encoding green fluorescent protein (GFP) were delivered to the HEK 293T cell line. After treating cells with lipoplexes, HG-U133 Affymetrix microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h 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.
  
  
  RESULTS
  Relative to untreated cells 2 h after lipoplex treatment, only downregulated genes were identified ≥ 30-fold: ALMS1, ITGB1, FCGR3A, DOCK10 and ZDDHC13. Subsequently, relative to GFP- cells, the GFP+ cell population showed at least a five-fold upregulation of RAP1A and PACSIN3 (8 h) or HSPA6 and RAP1A (16 and 24 h). Pharmacologic studies altering endogenous levels for ALMS1, FCGR3A, and DOCK10 (involved in filopodia protrusions), ITGB1 (integrin signaling), ZDDHC13 (membrane trafficking) and PACSIN3 (proteolytic shedding of membrane receptors) were able to increase or decrease transgene production.
  
  
  CONCLUSIONS
  RAP1A, PACSIN3 and HSPA6 may help lipoplex-treated cells overcome a transcriptional shutdown due to treatment with lipoplexes and provide new targets for investigating molecular mechanisms of transfection or for enhancing transfection through cell priming or engineering of the nonviral gene delivery system.
authors: Department of Pharmaceutical Sciences, Durham Research Center II, University of Nebraska-Medical Center, Omaha, NE, USA.
authors: Timothy M
authors: TM
authors: Martin
authors: Sarah A
authors: SA
authors: Plautz
authors: Angela K
authors: AK
authors: Pannier
mesh_terms: D019295:Computational Biology / Q000379:methods; D015870:Gene Expression; D020869:Gene Expression Profiling; D063990:Gene Ontology; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008055:Lipids; D010957:Plasmids / Q000235:genetics; D013997:Time Factors; D059467:Transcriptome; D014162:Transfection
chemical_list: D008055:Lipids; C086724:Lipofectamine
keywords: ALMS1; FCGR3A; GFP; HEK 293T; ITGB1; microarray analysis; nonviral gene delivery; temporal gene expression profile