Date of Award
Doctor of Philosophy
Gary S. Sayler
Michael L. Simpson, Steven W. Wilhelm, Todd B. Reynolds
Vertically aligned carbon nanofiber (VACNF) arrays have been developed as a novel tool for direct physical introduction and expression of DNA in mammalian cells (termed impalefection). This study describes the optimization of impalefection, the quantification of immobilized DNA on VACNFs, and the application of VACNFs in analysing gene expression in mammalian cells. Mechanical, chemical and biological parameters were optimized for impalefection. Alterations in a majority of the parameters resulted in no significant difference in impalefection efficiency, including nanofiber composition, DNA precipitation, cell confluency, cell concentration and sodium butyrate. The optimal DNA concentration ranged between 100 nanograms and 1 microgram, and the optimal impalefection substrate proved to be a Durx filter pad on plastic surface. High levels of efficiency in a wide range of mammalian cell lines demonstrated the versatile applicability of the impalefection method. Polymerase chain reaction (PCR) and in-vitro transcription (IVT) were used to investigate the transcriptional accessibility of immobilized DNA on VACNF arrays by correlating the yields of both IVT and PCR to that of non-immobilized DNA. Quantitative PCR was used to quantify the number of accessible yfp reporter gene copies immobilized to nanofiber arrays. DNA yields decreased dramatically in the non-immobilized control over time, while the majority of immobilized DNA was retained on VACNF arrays. These data demonstrated the development of methods for monitoring DNA immobilization techniques. To validate the applicability of VACNF arrays for controlling and monitoring mammalian gene expression, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and was impalefected into mammalian cells. VACNF arrays provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% ± 10.4% of impalefected cells were fully silenced by the inducible shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enabled rapid analysis of gene silencing effects using RNAi in single cells and cell populations.
Mann, David George James, "Vertically Aligned Carbon Nanofiber Arrays as a Platform for Gene Delivery and Expression Analysis in Mammalian Cells. " PhD diss., University of Tennessee, 2008.