(251d) Microarray Analysis of Gene Expression Profiles Characteristic of Successfully Transfected Cells | AIChE

(251d) Microarray Analysis of Gene Expression Profiles Characteristic of Successfully Transfected Cells


Riethoven, J. M. - Presenter, University of Nebraska-Lincoln

The design of efficient nonviral gene delivery systems offers potential uses in therapeutic applications such as tissue engineering, biomaterials, and gene therapy.  Nonviral gene delivery involves the delivery of exogenous gene(s) to cells, facilitated by the electrostatic complexation of the gene (as plasmid DNA) with a nonviral vector such as a cationic polymer or lipid.   Unlike viral based delivery systems, nonviral gene delivery systems can carry a larger genetic payload, overcome several safety concerns, and are easily modified to allow for cellular targeting.  However, current nonviral gene delivery systems are inefficient, prohibiting their use in therapeutic applications.  Much emphasis has been placed on increasing transfection efficiency through the physiochemical modification of the delivery vector.  While this empirical approach has resulted in some improvements, transfection efficiency has not been increased to levels desired.  Our research focuses on enhancing transfection through the identification and exploitation of endogenous intracellular mechanisms characteristic of successfully transfected cells.  We transfected HEK293T cells with a plasmid encoding EGFP and luciferase complexed with 25 kDa branched polyethyleneimine (PEI) and then separated successfully transfected cells (those expressing the green fluorescent protein, GFP+) from untransfected cells (those not expressing the green fluorescent protein, GFP-) using flow cytometry.  Subsequently, cDNA microarrays were used to identify the endogenous gene profile differences among GFP+, GFP-, and control cells using traditional cutoff limits of expression levels (greater than two fold difference in expression between data sets and False Discovery Rate (FDR) adjusted p-value less than 0.05).  Comparing gene profiles between control cells and GFP- cells revealed nine coding genes  to be differentially expressed, including RAP1A, HIST1H2AM, KRTAP19-1, HIST1H3H, HIST1H2AC, HIST2H4A, HIST1H2BD, SLC16A6, and EGR1 .  Comparing gene profiles between GFP+ cells and control cells revealed 405 coding genes to be differentially expressed.  Finally, comparing gene profiles between GFP+ cells and GFP- cells revealed 113 coding genes to be differentially expressed.  To better understand why one cell transfects (GFP+) while another cell does not (GFP-), as both groups of cells are treated identically in the same population, these 113 genes were further evaluated using Exploratory Gene Association Networks (EGAN) analysis, which revealed the majority (40.7 %) of the genes to be implicated in highly enriched Gene Ontology (GO) Processes of apoptosis, cell cycle signaling, cell response to stress, cytoskeletal signaling, DNA/RNA maintenance, motility, and transport.  Among the 113 genes, eight key genes (RAP1A, ACRC, ATF3, SCG5, ACTA1, MYH3, DDIT3, and PLK2) exhibited three-fold greater expression in GFP+ versus GFP- cells and were implicated in the identified GO pathways and five key genes (CHORDC1, WDR78, IREB2, PGAP1, NEB) were highly expressed in GFP+ versus GFP- cells (greater than fivefold upregulated).  Together, these 13 key genes were then further studied for their role in transfection using chemical and molecular activators and inhibitors of each gene pathway.  The concomitant elucidation of intracellular signaling pathways implicated in successful nonviral gene delivery combined with alteration of the endogenous gene profile as an adjuvant for delivery offers a novel approach to increase gene transfer for therapeutic applications.