Application of Genomic Technologies to Study Infertility Nicholas Rui Yuan Ho Washington University in St

Application of Genomic Technologies to Study Infertility Nicholas Rui Yuan Ho Washington University in St

Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Spring 5-15-2016 Application of Genomic Technologies to Study Infertility Nicholas Rui Yuan Ho Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Bioinformatics Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Yuan Ho, Nicholas Rui, "Application of Genomic Technologies to Study Infertility" (2016). Arts & Sciences Electronic Theses and Dissertations. 786. https://openscholarship.wustl.edu/art_sci_etds/786 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Division of Biology and Biomedical Sciences Computational and Systems Biology Dissertation Examination Committee: Donald Conrad, Chair Barak Cohen Joseph Dougherty John Edwards Liang Ma Application of Genomic Technologies to Study Infertility by Nicholas Rui Yuan Ho A dissertation presented to the Graduate School of Arts & Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2016 St. Louis, Missouri © 2016, Nicholas Rui Yuan Ho Table of Contents List of Figures ............................................................................................................................... v List of Tables .............................................................................................................................. vii Acknowledgements .................................................................................................................... viii Abstract ........................................................................................................................................ iv Introduction: Application of Genomic Technologies to Study Infertility .................................... 1 0.0 Title Page ............................................................................................................................ 1 0.1 Introduction Body .............................................................................................................. 2 0.2 References .......................................................................................................................... 4 Chapter 1: Computational Fertility Gene Candidate Identification .............................................. 5 1.0 Title Page ............................................................................................................................ 5 1.1 Introduction ........................................................................................................................ 6 1.1 Results ................................................................................................................................ 9 1.1.1 Mouse Prediction Model ........................................................................................ 10 1.1.2 Human Prediction Model ........................................................................................ 12 1.1.3 Model Predictions ................................................................................................... 15 1.1.4 Using Model Predictions in Studying Human Fertility Associated CNVs ............. 15 1.2 Discussion ......................................................................................................................... 16 1.3 Methods ............................................................................................................................. 22 1.3.1 Training Gene Sets .................................................................................................. 22 1.3.2 Gene Similarity Features......................................................................................... 24 1.3.3 Linear Discriminant Analysis Model ...................................................................... 25 1.3.4 Human Infertility Gene Deletion Analysis ............................................................. 26 1.4 Supplemental Figures ........................................................................................................ 28 ii 1.5 References ......................................................................................................................... 50 Chapter 2: Experimental in vivo Genetics Screen for Spermatogenesis Function ..................... 52 2.0 Title Page ........................................................................................................................... 52 2.1 Introduction ....................................................................................................................... 53 2.2 Results ............................................................................................................................... 58 2.2.1 Pilot shRNA Screen ............................................................................................... 58 2.2.2 Performance Benchmarks ....................................................................................... 63 2.2.1 Predicted Genes’ shRNA Screen ............................................................................ 65 2.3 Discussion ......................................................................................................................... 69 2.4 Methods ............................................................................................................................. 73 2.4.1 Gene Selection ....................................................................................................... 73 2.4.2 shRNA Pool Preparation ........................................................................................ 73 2.4.3 Mouse Testis Transfection ..................................................................................... 74 2.4.4 Cell Line Transfection ........................................................................................... 74 2.4.5 Illumina Sequencing Library Preparation .............................................................. 74 2.4.6 Statistical Analysis ................................................................................................. 75 2.4.7 Network Analysis ................................................................................................... 76 2.5 Supplemental Protocols, Figures, and Tables ................................................................... 77 2.5.1 Supplementary Protocol 2.1: Mouse Testis Transfection ...................................... 77 2.5.2 Supplementary Protocol 2.2: shRNA Pool DNA Preparation (For Injection) ....... 78 2.5.2 Supplementary Protocol 2.2: shRNA Pool Sequencing Library Preparation ........ 80 2.5.2 Supplementary Tables and Figures ........................................................................ 81 2.6 References ......................................................................................................................... 92 Chapter 3: Genetic Engineering to Rescue Fertility .................................................................. 94 3.0 Title Page ........................................................................................................................... 94 iii 3.1 Introduction ....................................................................................................................... 95 3.2 Results ............................................................................................................................... 96 3.2.1 Transgene Delivery ................................................................................................ 96 3.2.2 Endogenous Gene Repair ........................................................................................ 98 3.3 Discussion ....................................................................................................................... 103 3.4 Methods ........................................................................................................................... 104 3.4.1 Plasmid Cloning ................................................................................................... 104 3.4.2 Cell Line Transfection ......................................................................................... 104 3.4.3 DNA Library Preparation .................................................................................... 105 3.4.4 Data Analysis ....................................................................................................... 105 3.5 Supplemental Data and Tables ........................................................................................ 107 3.5 References ....................................................................................................................... 114 Summary: Application of Genomic Technologies to Study Infertility ..................................... 115 4.0 Title Page ......................................................................................................................... 115 4.1 Summary Body ...............................................................................................................

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