Microarray Time Course Assays of Cho-Cells Impaled on Vertically Arrayed Nanofibers

Abstract

Cells impaled onto nanofibers may be used as gene delivery devices to regulate expression of impaled cells, and to influence the cell-cell interactions of surrounding cells. To use this novel technology, it is essential to identify a time point following impalement where exogenous gene expression can be precisely monitored. RNA was extracted from CHO-cells impaled on vertically array nanofibers isolated over a series of time points ending at 24 hours. After fluorescent labeling, the RNA is hybridized to mouse microarray slides. Differential expression data was analyzed using Array Studio from Omicsoft. Functional annotation of the differentially expressed genes was performed using bioinformatics software including Genesis from Graz University of Technology, KEGG from Kanehisa Laboratories, and Web Gestalt from Vanderbilt University. Previous observations of membrane repair in response to wounding included reorganization of cytoskeleton and deployment of vesicles to the plasma membrane. Our data suggested that these mechanisms persisted up to four hours following impalement and membrane perturbation. Novel observations made were constant glycan activity, heparin sulfate, as a potential sealing factor, and over expression of G-protein coupled receptors labeled as Olfactory receptors. Surprisingly, induction of apoptosis was not detected during 24 hours. Finally, all observations show that by 24 hours differential expression has returned to nominal levels in comparison to control. This final key observation resolves a past criticism of a potentially very noisy expression background when conducting transfection experiments using plasmid linked nanofibers as the delivery method.