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Vertically Aligned Carbon Nanofiber Arrays As a Platform for Gene Delivery and Expression Analysis in Mammalian Cells

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Vertically Aligned Carbon Nanofiber Arrays As a Platform for Gene Delivery and Expression Analysis in Mammalian Cells University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2008 Vertically Aligned Carbon Nanofiber Arrays as a Platform for Gene Delivery and Expression Analysis in Mammalian Cells David George James Mann University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Microbiology Commons Recommended Citation 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. https://trace.tennessee.edu/utk_graddiss/362 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by David George James Mann entitled "Vertically Aligned Carbon Nanofiber Arrays as a Platform for Gene Delivery and Expression Analysis in Mammalian Cells." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Microbiology. Gary S. Sayler, Major Professor We have read this dissertation and recommend its acceptance: Michael L. Simpson, Steven W. Wilhelm, Todd B. Reynolds Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by David George James Mann entitled “Vertically Aligned Carbon Nanofiber Arrays as a Platform for Gene Delivery and Expression Analysis in Mammalian Cells.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Microbiology. __________________________ Gary S. Sayler, Major Professor We have read this dissertation and recommend its acceptance: ____________________________ Michael L. Simpson ____________________________ Steven W. Wilhelm ____________________________ Todd B. Reynolds Accepted for the Council: ______________________________ Carolyn R. Hodges, Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) VERTICALLY ALIGNED CARBON NANOFIBER ARRAYS AS A PLATFORM FOR GENE DELIVERY AND EXPRESSION ANALYSIS IN MAMMALIAN CELLS A Dissertation Presented for the Doctor of Philosophy Degree University of Tennessee, Knoxville DAVID GEORGE JAMES MANN May 2008 DEDICATION I dedicate this dissertation to my wife and best friend, Laura, who has lovingly and patiently stood by me through it all. The tapestry gets more and more beautiful every day. ii ACKNOWLEDGEMENTS I would first like to thank my mentor on this project, Tim McKnight, for his seemingly limitless supply of ideas and for his ambition and energy. He has taught me to go beyond the boundary of specific disciplines and more importantly, that “there is always time for one more experiment.” I want to thank my major professor, Dr. Gary Sayler, for his insight and guidance throughout this project and for teaching me to step into the role of an independent scientist. I would additionally like to thank the other members of my committee, Dr. Michael Simpson, Dr. Steven Wilhelm and Dr. Todd Reynolds for their continual support and input. I would like to thank Jack McPherson, the molecular biology wizard, who taught me much of what I know at the bench. His style of encouragement will always make me smile and not soon be forgotten. I want to thank Dr. Guy Griffin, who has the most respectable cell culture technique I have ever observed and who took the time to train me on culturing the CHOsen ones in between stories of the Lost Dutchman Mine. iii I would like to thank all my coworkers at the CEB over the years for their friendship. Memories from the graduate office will not soon be forgotten. I want to thank my family for their love and support and for encouraging me to pursue the biological sciences and to always set high goals. And most importantly, I would like to thank my wife Laura for her joy, patience and comfort throughout this project. Thanks for holding my hand all along the way. iv ABSTRACT 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 v 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. vi TABLE OF CONTENTS CHAPTER I........................................................................................................................ 1 LITERATURE REVIEW ............................................................................................... 1 Background................................................................................................................. 1 What is Nanotechnology?........................................................................................... 1 Nanotoxicology: An Emerging Field......................................................................... 4 The Promise of Nanotechnology in the Biological Realm ......................................... 8 Vertically Aligned Carbon Nanofibers as a Tool of Nanobiotechnology................. 12 Methods and Chemistry of Covalent DNA Immobilization..................................... 15 Traditional Methods of Gene Delivery in Mammalian Cells ................................... 21 Impalefection: Vertically Aligned Carbon Nanofiber-Mediated Gene Delivery .... 24 Potential Mammalian Cell Responses to Impalefection ........................................... 31 Inducible Gene Silencing and the RNA Interference Pathway................................. 33 OBJECTIVES OF THIS STUDY................................................................................. 37 CHAPTER II..................................................................................................................... 39 OPTIMIZATION OF THE VERTICALLY-ALIGNED CARBON NANOFIBER- MEDIATED TRANSFECTION METHOD IN MAMMALIAN CELLS ................... 39 Introduction............................................................................................................... 39 Materials and Methods ............................................................................................. 43 Plasmid isolation and maintenance........................................................................... 43 Cell culture and reagents........................................................................................... 43 Vertically aligned nanofiber-mediated impalefection .............................................. 44 Optimization of impalefection substrate................................................................... 45 Optimization of cell confluency................................................................................ 45 Optimization of DNA precipitation and concentration............................................. 46 Optimization of Cell Concentration
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