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Understanding the Role of Macf1 in the Hair Cell by Conditional Gene

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Understanding the Role of Macf1 in the Hair Cell by Conditional Gene UNDERSTANDING THE ROLE OF MACF1 IN THE HAIR CELL BY CONDITIONAL GENE TARGETING IN MICE AND CHARACTERIZING THE LOCALIZATION PATTERN OF DEMATIN by SHENYU SUN Submitted in partial fulfillment of the requirements for the degree of Master of Science Thesis Advisor: Brian M. McDermott Jr., Ph.D. Department of Biology CASE WESTERN RESERVE UNIVERSITY August, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Shenyu Sun candidate for the degree of Master of Science degree*. Committed Chair Jessica Fox, Ph.D. Committee Member Brian M. McDermott, Ph.D. Ruben Stepanyan, Ph.D. Deborah Harris, M.S. Date of Defense 06/05/2018 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents Acknowledgments ............................................................................................................................. 5 List of Tables ...................................................................................................................................... 6 List of Figures ..................................................................................................................................... 7 List of Abbreviations .......................................................................................................................... 8 Abstract ........................................................................................................................................... 10 Chapter 1: Background Introduction ................................................................................................ 11 1. Introduction ................................................................................................................................. 12 1.1 Hearing in the vertebrates ............................................................................................................ 13 1.2 Stereocilia ..................................................................................................................................... 19 1.3 Cuticular plate .............................................................................................................................. 20 1.4 Proteinaceous composition of the cuticular plate ....................................................................... 22 Chapter 2: Understanding the role of MACF1 in hair cell development by conditional gene targeting in mice ............................................................................................................................................. 27 2.1 Introduction .................................................................................................................................. 28 2.1.1 Introduction of MACF1 .......................................................................................................... 28 2.1.2 Introduction to Cre-loxP system ............................................................................................ 33 2.2 Materials and Methods ................................................................................................................ 35 2.2.1 Animals .................................................................................................................................. 35 2.2.2 Genotyping ............................................................................................................................ 35 2.3 Results .......................................................................................................................................... 38 2.4 Discussion ..................................................................................................................................... 44 Chapter 3: Identifying the expression pattern of Dematin, an actin-binding protein, in mouse inner ear ................................................................................................................................................... 46 3 3.1 Introduction .................................................................................................................................. 47 3.2 Materials and Methods ................................................................................................................ 48 3.2.1 Animals .................................................................................................................................. 48 3.2.2 Whole-mount immunolabeling ............................................................................................. 48 3.2.3 Imaging .................................................................................................................................. 49 4.3 Results .......................................................................................................................................... 49 4.4 Discussion ..................................................................................................................................... 52 References ....................................................................................................................................... 54 4 Acknowledgments First and foremost, I would like to thank my advisor, Dr. Brian McDermott for his continuous support and direction. In the past three years, he encouraged me to become a professional biologist and created an exciting research environment in the lab to pursue the truth of science. I would also like to thank the rest of my thesis committee: Dr. Jessica Fox, Dr. Ruben Stepanyan and M.S. Deborah Harris for their valuable support and guidance during my graduation. I am also very thankful to Dr. Ruben Stepanyan and Dr. Deborah Harris for their help with my experimental design and future career. I am very grateful to all of the members of the McDermott lab, past and present. Special thanks to Lana Pollock for her patient guidance in my first project. I would like to extend special thanks to Dr. Ahlam Salameh who helped me a lot in the Macf1 projects and Shaoyuan Zhu who taught me a lot in imaging. Thanks also to Caro Fernando, Hoa Nguyen, Zongwei Chen, Shenxuan Wang, Haimeng Bai, Robin Woods Davis, Kayla Kindig and Michael Dercoli for all their valuable advice and training. I am also thankful to Dr. Kumar Alagramam, Dr. Suhasini Gopal, Daniel Chen, Dr. Qing Yin Zheng and Dr. Martin Basch.for all of their help and for being great lab neighbors over the three years. In the end, I especially thankful to my parents for their great love and support over the three years, forming the strongest backup force on my way pursuing the beauty of science. 5 List of Tables Table 2.1 Primers used in Macf1 genotyping .................................................................................. 36 Table 2.2 PCR protocols used in Macf1 and Pax2-Cre genotyping .................................................. 37 Table 2.3 The Punnett square showing the predictdresults of the mouse genetic cross: (Female) Macf flox/+ X Macf1+/-; Pax2-Cre+/- (Male) ........................................................................... 43 6 List of Figures Figure 1.1 The components and structure of the human ear ......................................................... 16 Figure 1.2 The structure of the human cochlea and the location of hearing hair cell .................... 17 Figure 1.3 The structure of a hair cell ............................................................................................. 18 Figure 1.4 The types of cross-linking proteins in the cuticular plate .............................................. 24 Figure 1.5 Electron microscope images show the existence of cross-linking proteins in the cuticular plate .......................................................................................................................... 26 Figure 2.1 The structure of microtubule and actin cross-linking factor 1 (MACF1) ........................ 30 Figure 2.2 The localization pattern of MACF1 in adult mouse vestibular hair cells ........................ 31 Figure 2.3 The evidence of the linker bridging microtubule and actin in the cuticular plate ......... 32 Figure 2.4 The mechanism of Cre-loxP deletion ............................................................................. 34 Figure 2.5 The location and direction of loxP sites in gene Macf1.................................................. 37 Figure 2.6 The major mouse genetic cross experiment to generate the Macf1flox/-; Pax2-Cre conditional knockout mic ........................................................................................................ 40 Figure 2.7 Genotyping the Macf1flox and Pax2-Cre gene ................................................................ 41 Figure 2.8 The electrophoresis gel genotyping results of Macf1 inner ear conditional knockout mice ......................................................................................................................................... 42 Figure 3.1 Expression of dematin in mouse inner hair cells............................................................ 50 Figure 3.2 Expression of dematin in mouse vestibular hair cells .................................................... 51 7 List of Abbreviations ABR auditory brainstem response ACF7 actin cross-linking family protein 7 BPAG1 bullous pemphigoid antigen 1 BSA bovine serum albumin cAMP adenosine monophosphate CCV clathrin-coated vesicles CH domain calponin-homology domain CNS central nervous
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