DEVELOPMENT OF TRANSGENIC LIVESTOCK WITH REDUCED MYOSTATIN EXPRESSION USING RNA INTERFERENCE A Dissertation by KIMBERLY JEAN TESSANNE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2009 Major Subject: Veterinary Physiology DEVELOPMENT OF TRANSGENIC LIVESTOCK WITH REDUCED MYOSTATIN EXPRESSION USING RNA INTERFERENCE A Dissertation by KIMBERLY JEAN TESSANNE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Mark Westhusin Committee Members, Charles Long Thomas Spencer Nancy Ing Head of Department, Glen Laine December 2009 Major Subject: Veterinary Physiology iii ABSTRACT Development of Transgenic Livestock with Reduced Myostatin Expression Using RNA Interference. (December 2009) Kimberly Jean Tessanne, B.S., The Ohio State University; M.S., University of Wisconsin-Madison Chair of Advisory Committee: Dr. Mark Westhusin RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and short interfering RNAs (siRNAs) have been extensively employed for manipulating gene expression in a wide range of species. The goal for this research was to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. This would demonstrate the utility of RNAi for reducing gene expression in large animal species. To successfully target the myostatin gene for reduction, siRNAs were designed to target the both the bovine and caprine myostatin mRNA sequence. These were then tested for effectiveness in vitro using both an HEK 293T cell line expressing caprine myostatin as well as adult bovine muscle cells. The most effective siRNA, GDF8-1026, was cloned into a lentiviral plasmid and used to transduce bovine fetal fibroblasts for somatic cell nuclear transfer cloning as well as perivitelline injection of in vitro produced bovine embryos. To date, seven pregnancies have been established using these two methods. iv Concern over the possibility of off-target effects associated with the expression of shRNAs in vivo prompted investigation into tissue-specific expression. Therefore, investigation into the use of a muscle-specific promoter to drive transgene expression was pursued. The bovine myogenin promoter and muscle creatine kinase (MCK) promoter were cloned into a lentiviral plasmid and evaluated in bovine fetal muscle cells and mouse C2C12 cells in vitro for their ability to drive GFP expression. Both promoters demonstrated an increase in GFP intensity at day nine of differentiation when compared to the nontransduced control. The retroviral basis of lentiviral plasmids has raised concern over the possible development of replication competent lentivirus (RCL). Therefore, analysis of tissues from recipients of lentivirus-treated embryos was performed to detect possible RCL. Tissues and blood serum were tested for RCL using p24 ELISA as well as qRT-PCR for the VSV-G gene. To date all tissues tested so far shown no evidence for RCL using these analyses. Analysis of offspring transgenic for an shRNA targeting myostatin will allow confirmation of RNAi as a useful tool for manipulating gene expression in large animal species. v DEDICATION This dissertation is dedicated to my family, without whom I would never have been able to make it to where I am today. My parents unending love and support have been vital throughout my educational career. I will be forever grateful for their encouragement during difficult times. My grandparents have contributed to my growth as a student and as a person and will always be in my heart. Thank you to all who have been the backbone of my growth as a student and as a person. vi ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Westhusin, and my committee members, Dr. Long, Dr. Spencer, and Dr. Ing, for their guidance and assistance throughout the course of my career here at Texas A&M. I would also like to thank all of the present and past members of the Reproductive Sciences Lab at Texas A&M University, who were invaluable in this work. In addition, thanks to all of my family and friends for their support during this research program. vii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES................................................................................................... ix LIST OF TABLES .................................................................................................... x CHAPTER I INTRODUCTION AND LITERATURE REVIEW............................ 1 Introduction .................................................................................... 1 RNA Interference ........................................................................... 2 Lentiviral Delivery of shRNAs ...................................................... 5 Off-target Effects of RNAi............................................................. 9 Large Animal Research Models..................................................... 12 Development of Transgenic Livestock .......................................... 15 Myostatin........................................................................................ 18 II IN VITRO SUPPRESSION OF MYOSTATIN.................................... 23 Introduction .................................................................................... 23 Materials and Methods ................................................................... 24 Results ............................................................................................ 34 Discussion ..................................................................................... 44 Conclusions .................................................................................... 48 III ANALYSIS OF MUSCLE-SPECIFIC PROMOTERS FOR LENTIVIRAL-MEDIATED shRNA EXPRESSION.......................... 50 Introduction .................................................................................... 50 Materials and Methods ................................................................... 51 Results ............................................................................................ 54 viii CHAPTER Page Discussion ...................................................................................... 59 Conclusions .................................................................................... 61 IV PRODUCTION OF TRANSGENIC ANIMALS WITH REDUCED MYOSTATIN EXPRESSION............................................................. 63 Introduction .................................................................................... 63 Materials and Methods ................................................................... 65 Results ........................................................................................... 68 Discussion ...................................................................................... 74 Conclusions ................................................................................... 78 V REPLICATION COMPETENT LENTIVIRUS (RCL) ANALYSIS IN RECIPIENT ANIMALS OF TRANSGENIC EMBRYOS PRODUCED BY LENTIVIRAL TRANSFER..................................... 80 Introduction .................................................................................... 80 Materials and Methods ................................................................... 82 Results ........................................................................................... 85 Discussion ..................................................................................... 90 Conclusions ................................................................................... 92 VI SUMMARY.......................................................................................... 93 REFERENCES.......................................................................................................... 96 APPENDIX A .......................................................................................................... 113 APPENDIX B .......................................................................................................... 115 VITA ......................................................................................................................... 116 ix LIST OF FIGURES FIGURE Page 1 HEK 293T cells expressing caprine myostatin as well as an shRNA targeting myostatin..................................................................................... 35 2 Suppression of myostatin mRNA in vitro.................................................. 36 3 Bovine primary muscle cells in culture...................................................... 38 4 Immunocytochemistry on bovine primary muscle cells............................. 39 5 Transduction of primary bovine fetal muscle cells .................................... 41 6 Myotube development in adult bovine