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University of Florida Thesis Or Dissertation Formatting NOVEL GENOME ENGINEERING STRATEGIES TO MODEL REPEAT EXPANSION DISEASES By RUAN OLIVEIRA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Ruan Oliveira To my parents, whose hard work and continuous support allowed me to obtain a doctoral degree ACKNOWLEDGMENTS First, I would like to thank my parents, Rosane and Rudimar Oliveira, for their unconditional love and uninterrupted support over the last 26 years. Their commitment to my education makes me prouder than my own graduate degree. Next, I would like to express my deepest gratitude to Andriel Fenner, whose ears endured my daily complaints about graduate school. His continued support kept me sane and his company eased the process of transitioning into adulthood (in progress). Also, I would like to thank Maria Seabra for crossing my path in 2010, when my sophomore version went to a conference in São Paulo and met this loud and contagious woman who was unable to stop talking about her experiences as a Ph.D. student at the University of Florida. If I did not meet Maria, I would have never heard of Gainesville. I would like to thank my Ph.D. mentor, Dr. Maurice Swanson, for giving me freedom to pursue my own ideas and trusting me. I also thank Maury for teaching me how to be a scientist and for his patience with my learning curve. I am grateful to Myrna Stenberg, who taught me discipline and offered me psychological support in the moments I needed the most. I would like to thank Amrutha Pattamatta, Belinda Pinto, Barbara Perez and Franjo Ivanković, for their continuous emotional support and friendship. I am also very grateful to Curtis Nutter, Jodi Bubenik and James Thomas, who assisted me in my research endeavors, especially during the last year. Additionally, I would like to thank past and current members from the Swanson lab (Konstantinos Charizanis, Mini Manchanda, Marianne Goodwin, Apoorva Mohan, Marina Scotti, Catherine Marten, Łukasz Sznajder, Olgert Bardhi, Jordan Ross, Katherine Narvaez and Jorge Cañas) for creating a pleasant work environment and for their help and/or advice. 4 I would like to thank the Graduate Program in Biomedical Sciences, especially Peggy Wallace and the Genetics concentration, for their commitment to graduate education. I am also grateful to my committee members, Laura Ranum, Eric Wang, Susan Semple-Rowland and Naohiro Terada, for their availability and guidance. Last, I thank the Department of Molecular Genetics and the Center for NeuroGenetics, for their resources and support in the last 4 years. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 8 LIST OF FIGURES .............................................................................................................. 9 LIST OF ABBREVIATIONS ............................................................................................... 11 ABSTRACT ........................................................................................................................ 15 CHAPTER 1 INTRODUCTION ........................................................................................................ 17 Repeat Expansion Diseases ...................................................................................... 17 Pathomechanisms of Repeat Expansions ................................................................. 18 Myotonic Dystrophy Type I: Mechanisms and Mouse Models .................................. 21 2 ENGINEERING THE ENDOGENOUS DMPK LOCUS ............................................. 31 Background ................................................................................................................. 31 Targeting the Endogenous Dmpk 3’ UTR by CRISPR/Cas9..................................... 34 Gibson Assembly of CTGexp Recombination Templates ........................................... 36 Outcomes of CRISPR/Cas9 Zygote Microinjections ................................................. 37 Synthesis of Recombination Templates Carrying Larger Repeat Expansions ......... 40 The Knockin of 480 CTG Repeats in the Endogenous Dmpk 3’ UTR ...................... 41 Discussion ................................................................................................................... 42 3 PHENOTYPIC OUTCOMES OF DMPK KNOCKIN MODELS .................................. 54 Background ................................................................................................................. 54 Transcriptional and Translational Assessments in Dmpk170 Muscles ....................... 55 Accumulation of RNA Foci in the Absence of Spliceopathy ...................................... 57 Increased Levels of RNA Foci Following Myogenic Differentiation ........................... 58 The Transcriptomic Profile of Dmpk170/170 Myotubes ................................................. 59 Discussion ................................................................................................................... 60 4 THE CHOROID PLEXUS INVOLVEMENT IN DM1 .................................................. 68 Background ................................................................................................................. 68 In the Dmpk170 Brain, RNA Foci Are Restricted to Choroidal Epithelial Cells ........... 69 Increased Dmpk Expression in the Mouse Choroid Plexus ...................................... 70 Choroid Plexus Development, Functions and Pathology .......................................... 71 Consequences of Mbnl Loss-of-Function in the Choroid Plexus .............................. 75 6 Discussion ................................................................................................................... 79 5 CONCLUSIONS AND FUTURE DIRECTIONS ......................................................... 90 6 MATERIALS AND METHODS.................................................................................... 94 Design and Validation of crRNAs Targeting Dmpk .................................................... 94 Propagation of Plasmids in Escherichia coli .............................................................. 95 Gibson Assembly of Recombination Templates ........................................................ 95 Rolling Circle Amplification ......................................................................................... 97 Repeat Dimerization by Golden Gate Assembly ....................................................... 98 Zygote Microinjections ................................................................................................ 98 Isolation of Genomic DNA from Tail Snips ................................................................. 99 Southern Blotting ......................................................................................................100 PCR Genotyping .......................................................................................................101 Repeat-primed PCR..................................................................................................101 RNA Isolation from Dissected Tissues and Cultured Cells .....................................102 cDNA Synthesis ........................................................................................................103 Quantitative PCR ......................................................................................................103 Splicing Analysis by RT-PCR ...................................................................................104 Western Blotting ........................................................................................................104 RNA Fluorescence in situ Hybridization ...................................................................105 Myoblast Isolation, Culture and Differentiation ........................................................106 RNA-Seq ...................................................................................................................106 LIST OF REFERENCES .................................................................................................108 BIOGRAPHICAL SKETCH ..............................................................................................123 7 LIST OF TABLES Table page 1-1 Mouse models developed to study and treat myotonic dystrophy ........................ 29 2-1 Primers used to amplify the targeted Dmpk locus for T7E1 assay ....................... 52 2-2 Primers used to amplify homology arms for Gibson assembly ............................. 52 2-3 Primers used to amplify Southern blot probes and resulting amplicons............... 52 2-4 Primers used to amplify the edited Dmpk 3’ UTR locus for genotyping purposes ................................................................................................................. 53 2-5 Primers used for fluorescent repeat-primed PCR ................................................. 53 2-6 Mutant Dmpk founders: germline transmission rates and expansion length ........ 53 3-1 Primers used to measure transcript levels by qPCR ............................................. 67 3-2 Primers used to asses Dmpk transcript distribution and alternative splicing by RT-PCRs................................................................................................................
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