TARGETED MUTAGENESIS OF ZEBRAFISH HEARING-RELATED GENES USING ZFN AND TALEN by LI LIU Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Advisor: Dr. Brian M. McDermott Department of Biology CASE WESTERN RESERVE UNIVERSITY January, 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Li Liu _________________________________________________________ Master of Science candidate for the __________________________________degree *. Jean H. Burns (signed)_________________________________________________ (chair of the committee) Brian M. McDermott __________________________________________________ Claudia Mizutani __________________________________________________ Emmitt R. Jolly __________________________________________________ __________________________________________________ __________________________________________________ 10/28/2013 (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein TABLE OF CONTENTS LIST OF FIGURES...............................................................................................................2 ACKNOWLEDGEMENT....................................................................................................3 LIST OF ABREVIATIONS.................................................................................................4 ABSTRACT............................................................................................................................5 INTRODUCTION.................................................................................................................6 Hearing and hair cells.........................................................................................6 Hearing related genes.......................................................................................11 Zebrafish as a model organism.....................................................................15 Gene targeting technology..............................................................................16 MATERIALS AND METHODS......................................................................................21 RESULTS.............................................................................................................................28 CoDA-ZFN induced somatic mutations in zebrafish fascin 2a gene in a site-directed manner.....................................................................28 CoDA-ZFN induced site-specific heritable mutations in zebrafish piezo 2-4 gene..................................................................................32 TALEN induced heritable mutations in zebrafish piezo 2-4 gene with a higher efficiency.........................................................................38 DISCUSSION.......................................................................................................................43 REFERENCES.....................................................................................................................48 1 LIST OF FIGURES FIGURE 1...............................................................................................................................7 FIGURE 2...............................................................................................................................8 FIGURE 3............................................................................................................................10 FIGURE 4.............................................................................................................................12 FIGURE 5.............................................................................................................................14 FIGURE 6.............................................................................................................................17 FIGURE 7.............................................................................................................................18 FIGURE 8.............................................................................................................................20 FIGURE 9.............................................................................................................................27 FIGURE 10..........................................................................................................................29 FIGURE 11..........................................................................................................................30 FIGURE 12..........................................................................................................................31 FIGURE 13..........................................................................................................................32 FIGURE 14..........................................................................................................................33 FIGURE 15..........................................................................................................................35 FIGURE 16..........................................................................................................................35 FIGURE 17..........................................................................................................................37 FIGURE 18..........................................................................................................................39 FIGURE 19..........................................................................................................................40 FIGURE 20..........................................................................................................................41 FIGURE 21..........................................................................................................................42 FIGURE 22..........................................................................................................................44 TABLE 1..............................................................................................................................22 TABLE 2..............................................................................................................................22 TABLE 3..............................................................................................................................30 TABLE 4..............................................................................................................................31 TABLE 5..............................................................................................................................34 TABLE 6..............................................................................................................................39 2 ACKNOWLEDGEMENT My first acknowledgement and deepest appreciation goes to my advisor Dr. Brian M. McDermott for his excellent guidance, patience and caring. He encouraged me to become not only an experimentalist, but also an independent thinker on research. He spent time answering my questions, as well as encouraging and motivating me to find out the answer on my own. He has been a great mentor as well as a thoughtful friend who cares about my life, my family and supports my long-term objectives. I also would like to express gratitude to the rest of my committee members, Dr. Claudia Mizutani and Dr. Emmitt R. Jolly for their encouragement, insightful comments, and hard questions. I greatly appreciate their time talking to me and responding to my questions. I would like to thank Shih-wei Chou, who as a good friend and a co-worker has been always willing to instruct and help since the first day I worked with her. Many thanks to Carol Fernando, Jiaqi Hu, and other people in the laboratory for supporting me in work and in daily life. My research would not been possible without them. I would like to thank the Department of Biology at Case Western Reserve University for the diverse resources and help they provided. Finally, my heartfelt appreciation goes to my parents, who have been supporting me psychologically and financially in the past 23 years. Their continuous love provided me courage to pursue my research at the other end of the world. 3 LIST OF ABBREVIATIONS NIDCD, national institute of deafness and other communication disorder; F-actin, filamentous actin; ZFN, zinc finger nuclease; TALEN, transcription activator-like nuclease; FSCN, fascin; PKC, protein kinase C; MA, mechanically activated; Dmpiezo, Drosophila Piezo; Mmpiezo, mouse Piezo; RNAi, RNA interference; MOs, morpholino oligos; ZF, zinc finger; NHEJ, non-homologous end joining; CoDA, context-dependent assembly; TAL, transcription activator-like; RVD, repeat variable di-residues; HRMA, high melting resolution analysis; dpf, day-post-fertilization; Tm, annealing temperature; sgRNAs, single guide RNAs 4 Targeted mutagenesis of zebrafish hearing-related genes using ZFN and TALEN Abstract by LI LIU Hair cells are specialized mechanosensory cells with stereocilia protruding from their apical surfaces. Deflection of stereocilia allows the opening of mechanotransduction channels on the tips of the stereocilia. However, regulation of stereocilia development at the molecular level and the identity of the mechanotransduction channel remain largely unknown. Engineered ZFN and TALEN have shown to be efficient genome editing tools among many different organisms.
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