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Analysis of Transciptome and Splicing Changes In ANALYSIS OF TRANSCIPTOME AND SPLICING CHANGES IN ZEBRAFISH MODEL FOR SPINAL MUSCULAR ATROPHY HIMANSHU VYAS NATIONAL UNIVERSITY OF SINGAPORE 2016 ANALYSIS OF TRANSCIPTOME AND SPLICING CHANGES IN ZEBRAFISH MODEL FOR SPINAL MUSCULAR ATROPHY HIMANSHU VYAS (M. Tech. Biotechnology, Indian Institute of Technology Guwahati) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2016 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have dully acknowledged all the sources of information which have been used in the thesis. This thesis has also not been used for any degree in any university previously. Himanshu Vyas January 2016 ACKNOWLEDGEMENT I would like to thank my supervisor, Assoc. Prof. Christoph Winkler, for his valuable guidance and supervision during the entire project. He has been patient with all the mistakes and confusions that accompanied me throughout my work. He has been a constant source of motivation and a driving force behind the success of this work. I would also like to thank all the members of Winkler lab, both former and present, for their support in last 4 years. I would like to specially thank Anita for all her help and support during RNAseq work. I am also very grateful to Manish, Husvinee, Angela, Sudha, Shijie, Zoltan, Tingsheng for all the friendly conversations and being there whenever I needed to discuss science. I want to thank Shermaine and Viktor for their help with the CRISPR work. I also very thankful to Mr. Balan and entire fish facility team for providing zebrafish embryos. I am obliged to Shermaine and Kelvin for providing me with motor neuron transgenic line and Schwann cell calcium sensor line, respectively. I owe earnest thankfulness to Dr. Candida Vaz for introducing me to the Bioinformatics and teaching me all the software. I also thank our collaborator Dr. S. Mathavan (GIS) for all his help with the RNAseq, Dr. Vivek Tanavde and Dr. Brian Parker (BII) for their help with Bioinformatic analysis of my data. I am grateful to our collaborator Dr. Sibylle Jablonka (University of Wuerzburg) for providing with IGHMBP2 MO and IGHMBP2 antibody for DSMA1 project. i I am grateful to all my friends in Singapore, especially Sarath, Manish, Digant and Dhiraj, who have been there with me since starting, be it holiday trips, night-outs or scientific and political discussions. Last but not least, I want to thank my wife and my parents for their unlimited love and support without which this was not possible. I wish to dedicate this work to them and my late grandfather, who has been my idol for as long as I can remember. Thank you. ii Table of contents ACKNOWLEDGEMENT .............................................................................................................. i PUBLICATIONS ........................................................................................................................ vi CONFERENCE CONTRIBUTIONS .......................................................................................... vii SUMMARY .............................................................................................................................. viii LIST OF FIGURES ..................................................................................................................... x LIST OF TABLES ...................................................................................................................... xi LIST OF ABBREVIATIONS ..................................................................................................... xii 1. INTRODUCTION .................................................................................................................... 1 1.1 Spinal Muscular Atrophy (SMA) ................................................................................... 1 1.1.1 Loss of Survival of Motor Neuron 1 (SMN1) causes Spinal Muscular Atrophy.. 1 1.1.2 Role of SMN in assembly of U snRNP complexes ............................................ 4 1.1.3 The role of SMN in motor axons ....................................................................... 8 1.1.4 Non-cell autonomous effects in SMA .............................................................. 10 1.2 The role of Schwann cells for maintenance of motor neurons ................................... 12 1.3 Distal Spinal Muscular Atrophy Type 1 (DSMA1) ....................................................... 15 1.4 RNA sequencing (RNAseq) ....................................................................................... 16 1.5 CRISPR/Cas system .................................................................................................. 18 1.6 Aim of the project ....................................................................................................... 20 2. MATERIAL AND METHODS ................................................................................................ 21 2.1 Zebrafish strains and handling ................................................................................... 21 2.2 Antisense morpholino oligonucleotide (MO) knockdown ............................................ 22 2.3 Whole-mount immunostaining .................................................................................... 22 2.4 Whole zebrafish embryo dissociation ......................................................................... 23 2.5 FACS ......................................................................................................................... 25 2.6 RNAseq library preparation ........................................................................................ 25 2.6.1 Total RNA extraction ....................................................................................... 26 2.6.2 Double stranded cDNA synthesis ................................................................... 26 2.6.3 Covaris shearing ............................................................................................. 28 2.6.4 Qubit assay ..................................................................................................... 28 2.6.5 cDNA library generation .................................................................................. 29 2.6.6 Quantitative PCR ............................................................................................ 30 2.7 RNAseq data analysis ................................................................................................ 31 2.8 Whole-mount in situ hybridisation .............................................................................. 32 2.8.1 Cloning of riboprobes for WISH ...................................................................... 32 2.8.2 Probe synthesis .............................................................................................. 33 2.8.3 In situ hybridization ......................................................................................... 34 2.9 DNA Sequencing........................................................................................................ 36 2.10 Agarose gel electrophoresis ..................................................................................... 36 iii 2.11 RNA extraction ......................................................................................................... 37 2.11.1 DNase I digestion .......................................................................................... 38 2.11.2 RNeasy clean-up .......................................................................................... 38 2.12 First strand cDNA synthesis for RT-PCR ................................................................. 39 2.13 Alternative splicing assay by semiquantitative RT-PCR ........................................... 39 2.14 General PCR ............................................................................................................ 40 2.15 Western Blot............................................................................................................. 40 2.16 CRISPR/Cas9 .......................................................................................................... 43 2.16.1 CRISPR target site design and gRNA preparation ........................................ 43 2.16.2 Cas9 mRNA preparation ............................................................................... 44 2.16.3 Microinjection ................................................................................................ 45 2.16.4 Genomic DNA (gDNA) extraction .................................................................. 46 2.16.5 srsf6b mutant screening ................................................................................ 46 3. RESULTS ............................................................................................................................. 47 3.1 A knockdown of Smn leads to motoaxonal defects in zebrafish embryos .................. 47 3.2 Generation of a zebrafish model for DSMA1 .............................................................. 48 3.2.1 An IGHMBP2 knockdown leads to motoaxonal defects similar to SMA .......... 48 3.3 HB9:eGFP/mCherry embryo dissociation and FAC sorting of motor neurons ............ 52 3.3.1 Optimization of embryo dissociation conditions and FAC sorting of motor neurons .................................................................................................................... 52 3.3.2 Total RNA extraction from sorted motor
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