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Characterization of Human Macroh2a Through Gene Targeting Shawn C Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2014 Characterization of Human MacroH2A Through Gene Targeting Shawn C. Moseley Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES CHARACTERIZATION OF HUMAN MACROH2A THROUGH GENE TARGETING By SHAWN C. MOSELEY A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science Degree Awarded: Spring Semester, 2014 Shawn C. Moseley defended this thesis on March 31, 2014. The members of the supervisory committee were: Brian Chadwick Professor Directing Thesis Jonathan Dennis Committee Member Karen McGinnis Committee Member The Graduate School has verified and approved the above-named committee members and certifies that the thesis has been approved in accordance with university requirements. ii ACKNOWLEDGMENTS I would like to thank my advisor Dr. Brian Chadwick, as well as Dr. Jonathan Dennis and Dr. Karen McGinnis for their advice, assistance, and support. I would also like to thank all of the members of the Chadwick lab, past and present, for their support. iii TABLE OF CONTENTS List of Tables ............................................................................................................................. vi List of Figures ...........................................................................................................................vii Abstract ..................................................................................................................................... ix INTRODUCTION AND BACKGROUND ................................................................................. 1 GENERATING RESOURCES FOR GENOME ENGINEERING ............................................... 3 Introduction ........................................................................................................................ 3 Generating Zinc Finger Nucleases (ZFNs) .......................................................................... 4 Generating Constructs ......................................................................................................... 8 Methods ............................................................................................................................ 11 TARGETING MACROH2A1 AND MACROH2A2 ................................................................. 13 Targeting MacroH2A1 ...................................................................................................... 13 Targeting MacroH2A2 ...................................................................................................... 18 Targeting MacroH2A2 with TALENs ............................................................................... 19 Retargeting MacroH2A1 ................................................................................................... 22 Methods ............................................................................................................................ 26 EXPRESSION ANALYSIS OF MACROH2A1 KNOCKOUTS ............................................... 30 Expression Analysis of Clone-8 ........................................................................................ 30 Expression Analysis of Clone-21 ...................................................................................... 35 Expression Analysis of Clone-64 ...................................................................................... 45 Methods ............................................................................................................................ 48 TRANSGENE RESCUE OF MACROH2A1............................................................................. 50 Generating Clone-21 Derived MacroH2A1 Rescues .......................................................... 50 Generating Clone-64 Derived MacroH2A1 Rescues .......................................................... 53 Methods ............................................................................................................................ 53 CHROMATIN ANALYSIS IN MACROH2A1 MUTANTS ..................................................... 57 Chromatin Immunoprecipitation of CCL2 ......................................................................... 57 Analysis of the Inactive X Chromosome by Immunofluorescence and FISH ..................... 58 Methods ............................................................................................................................ 62 CONCLUSIONS AND DISCUSSION ...................................................................................... 70 APPENDIX .............................................................................................................................. 72 iv A. PRIMERS ......................................................................................................................... 72 REFERENCES ......................................................................................................................... 75 BIOGRAPHICAL SKETCH ..................................................................................................... 79 v LIST OF TABLES 1 Genes of Interest – Set 1. ............................................................................................... 32 2 Summary of Quantitative RT-PCR Trends for MacroH2A1 Knockouts Compared to RNAseq ......................................................................................................................... 34 3 Genes of Interest – Set 2 ................................................................................................ 34 4 Summary of Quantitative RT-PCR Trends for all MacroH2A1 Targets Compared to RNAseq ......................................................................................................................... 37 5 Genes of Interest – Set 3 ................................................................................................ 37 6 Summary of Quantitative RT-PCR Trends for MacroH2A1 Clone-21 Compared to Microarray. .................................................................................................................... 38 7 Genes of Interest – Set 4 ................................................................................................ 39 8 Summary of RT-PCR Trends for Gene Set 4 ................................................................. 39 9 Gene Expression Changes Comparing Different Passage Controls to Clone-21 .............. 41 10 Genes of Interest – Set 5 ................................................................................................ 42 11 Summary of RT-PCR Trends for Gene Set 5 ................................................................. 43 12 Genes of Interest – Set 6 ................................................................................................ 44 vi LIST OF FIGURES 1 MacroH2A ZFN Luciferase Assays ................................................................................. 6 2 MacroH2A2 ZFN T7EI Assay ......................................................................................... 7 3 MacroH2A1 (Site 2) Homology Mediated Repair Assay .................................................. 8 4 Structure of the Synthetic Exon Promoter Trap Plasmid (pSEPT) .................................... 9 5 Schematic Illustration of the MacroH2A1 Targeting Strategy ........................................ 10 6 MacroH2A1 Targeting Screen ....................................................................................... 14 7 Sequence Analysis of NHEJ Induced Mutation of MacroH2A1 Exon-2 ......................... 15 8 Anti-macroH2A1 Immunofluorescence ......................................................................... 16 9 Western Blot Analysis of MacroH2A1 Knockouts ......................................................... 17 10 Quantitative RT-PCR Analysis of MacroH2A1 Knockouts ............................................ 17 11 Potential MacroH2A2 Exon-2 Start Sites ....................................................................... 19 12 MacroH2A2 TALEN FLASH Assembly ....................................................................... 20 13 MacroH2A2 TALEN Cut Assays................................................................................... 21 14 MacroH2A1 Retargeting Screen .................................................................................... 22 15 Quantitative RT-PCR Analysis of New MacroH2A1 Knockout ..................................... 23 16 Additional Western Blots of MacroH2A1 Knockouts .................................................... 24 17 Anti-macroH2A2 Immunofluorescence ......................................................................... 25 18 MacroH2A1 Sequence Alignment ................................................................................. 30 19 Scatter Plot of Gene Expression Changes in MacroH2A1 Knockout .............................. 31 20 Quantitative RT-PCR Analysis on Genes of Interest – Set 1 .......................................... 33 21 Quantitative RT-PCR Analysis on Genes of Interest – Set 2 .......................................... 36 22 Quantitative RT-PCR Analysis on Genes of Interest – Set 3 .......................................... 38 vii 23 RT-PCR Analysis on Genes of Interest – Set 4 .............................................................. 39 24
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