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Dissertation Final (Shuang Liang) NEW APPROACH TO DNA TRANSFECTION AND GENETICS IN SCHISTOSOME PARASITES By SHUANG LIANG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Emmitt R. Jolly Department of Biology CASE WESTERN RESERVE UNIVERSITY May 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Shuang Liang candidate for the degree of Doctor of Philosophy*. Committee Chair Hillel J. Chiel Committee Member Emmitt R. Jolly Committee Member Brian M. McDermott Committee Member Claudia Mizutani Committee Member Pieter L. deHaseth *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright©2015 by Shuang Liang All rights reserved ii Thesis Outline Acknowledgements Table of contents Abstract Chapter 1: Introduction and significance Chapter 2: Polyethyleneimine mediated DNA transfection in schistosome parasites and regulation of the WNT signaling pathway by a dominant-negative SmMef2 Chapter 3: Evaluation of schistosome promoter expression for transgenesis and genetic analysis Chapter 4: Discussions and future directions Appendix: Molecular cloning and characterization of Hsp90 in the crayfish P. clarkii Bibliography i Acknowledgements This is a wide spread picture on the Internet drawn by a professor from University of Utah. The figure illustrates exactly how a tiny breakthrough off the boundary of the circle from a Ph.D. project contributes to the world. During my six-year school life at Case Western Reserve University, my thesis advisor Dr. Emmitt Jolly set a role model to keep reminding me how to push further from the boundary you are focused on as well as keep your eyes open to the whole universe. I would like to take this opportunity to gratefully acknowledge the generous inspirations and unflagging encouragement from Dr. Jolly. I would also like to thank the members of my Ph.D. committee, Professors Pieter deHaseth, Claudia Mizutani, Brian McDermott and Hillel Chiel for their helpful suggestions, guidance and supports in general. ii Finally, I would like to thank my family for their patience and understanding. I am forever indebt to them. iii Table of Contents Thesis Outline .................................................................................................. i Acknowledgements ........................................................................................ ii Table of Contents .......................................................................................... iv List of Tables ................................................................................................ viii List of Figures ................................................................................................ ix Abstract ........................................................................................................... 1 Chapter I: Introduction and Significance ..................................................... 3 1.1 Introduction ........................................................................................... 3 1.2 Geographical distribution of schistosomiasis ................................... 4 1.3 Schistosome Lifecycle ......................................................................... 5 1.4 Pathogenesis ......................................................................................... 7 1.5 Diagnosis of Schistosomiasis ........................................................... 10 1.6 Treatment ............................................................................................. 11 1.7 The cercariae/ schistosomula transition .......................................... 12 1.7.1 Morphology ..................................................................................... 13 1.7.2 In vitro transformation and cultivation of schistosomula ................. 15 1.7.3 Schistosome genomics and transcriptomics .................................. 16 1.8 Schistosome transfection .................................................................. 17 1.9 Polyethyleneimine transfection ......................................................... 23 1.10 Thesis objectives .............................................................................. 23 iv Chapter II: Polyethyleneimine Mediated DNA Transfection in Schistosome Parasites and Regulation of the WNT Signaling Pathway by a Dominant-Negative SmMef2 ..................................................................... 24 2.1 Abstract................................................................................................ 25 2.2 Introduction ......................................................................................... 25 2.3 Materials and methods ....................................................................... 29 2.3.1 Animal preparation ......................................................................... 29 2.3.2 Construction of vectors for transfection .......................................... 30 2.3.3 PEI-mediated plasmid transfection ................................................. 33 2.3.4 Total DNA/RNA extraction from schistosomula .............................. 34 2.3.5 Transfection and gene transcript analysis ...................................... 34 2.3.6 Protein expression and western blot analysis ................................ 37 2.4 Results ................................................................................................. 38 2.4.1 PEI facilitates the transfection of DNA in schistosomes ................. 38 2.4.2 CMV and Sm23 promoters can induce gene expression from a plasmid in PEI transfected schistosomula ............................................... 41 2.4.3 Schistosome Mef2 is transcriptionally autoregulated and regulates WNT gene transcript levels ..................................................................... 44 2.4.4 SmMef2 protein is expressed in schistosomes .............................. 49 2.5 Discussion ........................................................................................... 51 2.6 Supporting information ...................................................................... 57 2.6.1 Supplemental figures ...................................................................... 57 2.6.2 Supplemental tables ....................................................................... 59 Chapter III: Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis ........................................................... 61 v 3.1 Abstract................................................................................................ 62 3.2 Introduction ......................................................................................... 63 3.3 Materials and methods ....................................................................... 65 3.3.1 Animals and parasites .................................................................... 65 3.3.2 DNA plasmid construction .............................................................. 66 3.3.3 PEI-mediated transfection .............................................................. 69 3.3.4 RNA extraction ............................................................................... 69 3.3.5 Gene transcript analysis ................................................................. 70 3.3.6 Protein extraction and western blot analysis .................................. 72 3.3.7 Schistosomula viability measurement ............................................ 72 3.3.8 Caspase activity assay ................................................................... 73 3.4 Results ................................................................................................. 74 3.4.1 Constitutive promoters can induce strong and weak gene expression in schistosomes .................................................................... 74 3.4.2 The SmActin1 promoter drives high levels of endogenous gene expression and can induce gene-specific lethality in schistosomes ........ 78 3.4.3 SmActin1, CMV and SmHsp70 promoters induce different levels of elevated SmCaspase7 transcript expression .......................................... 84 3.4.4 Overexpression of SmMef2 under different vitality of promoters lead to distinct phenotypes .............................................................................. 92 3.5 Discussion ........................................................................................... 93 3.6 Supporting information ...................................................................... 98 Chapter IV: Discussion and Future Directions ........................................ 101 4.1 Stable transfection of schistosomes .............................................. 101 4.2 Determination of transfection efficiency ........................................ 103 vi 4.3 Applications of PEI transfection in schistosome ........................... 105 Appendix: Molecular cloning and characterization of Hsp90 in the crayfish P. clarkii ........................................................................................ 107 5.1 Abstract.............................................................................................. 108 5.2 Introduction ....................................................................................... 108 5.3 Material and methods ....................................................................... 112 5.3.1 Animals ........................................................................................
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