MASTERARBEIT Titel der Masterarbeit A zebrafish model of Letm1 and Letm2 deficiency verfasst von Stefan Hajny, BSc angestrebter akademischer Grad Master of Science (MSc) Wien, 2015 Studienkennzahl lt. Studienblatt: A 066 834 Studienrichtung lt. Studienblatt: Masterstudium Molekulare Biologie (UG2002) Betreut von: Priv. Doz. Dr. Karin Novikovsky Table of contents Acknowledgements ............................................................................................................... I Abbreviations ...................................................................................................................... II 1. Introduction .................................................................................................................... 1 1.1. Wolf Hirschhorn Syndrome .............................................................................................................................. 1 1.2. LETM1, an important protein for mitochondrial potassium homeostasis ....................................................... 1 1.3. The KHE is an indispensable complex to maintain mitochondrial volume and pH homeostasis ..................... 3 1.4. The elusive role of LETM2, a protein closely related to LETM1 ....................................................................... 5 1.5. The zebrafish as a model organism .................................................................................................................. 6 1.6. Genomic engineering approaches ................................................................................................................... 7 1.6.1 TALEN ......................................................................................................................................................... 7 1.6.2 The CRISPR/Cas9 system ............................................................................................................................ 9 1.6.3 Multisite Gateway Cloning ....................................................................................................................... 10 1.7. Studying different aspects of LETM1 and LETM2 in vivo and in vitro ............................................................ 13 2. Material and Methods .................................................................................................... 14 2.1. Animal handling ............................................................................................................................................. 14 2.1.1 Husbandry ................................................................................................................................................ 14 2.1.2 Breeding and nutrition ............................................................................................................................. 14 2.1.3 Microinjections......................................................................................................................................... 14 2.1.4 Genotyping ............................................................................................................................................... 15 2.1.5 Euthanasia ............................................................................................................................................... 16 2.2. General procedures ....................................................................................................................................... 17 2.2.1 Restriction digest ..................................................................................................................................... 17 2.2.2 Agarose gel electrophoresis ..................................................................................................................... 17 2.2.3 Agarose gel extraction ............................................................................................................................. 17 2.2.4 Bacterial transformation and Inoculation ................................................................................................ 17 2.2.5 DNA Isolation ........................................................................................................................................... 18 2.2.6 RNA isolation and cDNA synthesis ........................................................................................................... 19 2.2.7 PCR Protocols and primer ........................................................................................................................ 20 2.2.8 Sequencing ............................................................................................................................................... 22 2.3. Genetic modifications .................................................................................................................................... 22 2.3.1 Gateway Cloning ...................................................................................................................................... 22 2.3.2 Generation of zebrafish, overexpressing letm1 or letm2 ......................................................................... 26 2.3.3 TALEN induced knock-out ........................................................................................................................ 26 2.3.4 TALEN mediated knock-in ........................................................................................................................ 31 2.3.5 CRISPR/Cas9 induced knock-out .............................................................................................................. 32 2.4. In situ hybridization ....................................................................................................................................... 36 2.4.1 Preparation of probes .............................................................................................................................. 36 2.4.2 Whole mount in situ hybridization ........................................................................................................... 37 2.4.3 Brain sections ........................................................................................................................................... 39 2.5. Quantitative gene expression analysis ........................................................................................................... 40 2.5.1 qPCR analysis ........................................................................................................................................... 40 2.6. Localization studies in HeLa cells ................................................................................................................... 41 2.7. Locomotor behavior ....................................................................................................................................... 41 2.8. Bioinformatical approaches and data evaluation .......................................................................................... 42 2.8.1 Phylogenetic analysis ............................................................................................................................... 42 2.8.2 Software ................................................................................................................................................... 44 2.9. Utilized buffers and commercial kits.............................................................................................................. 45 3. Results ........................................................................................................................... 48 3.1. The gene duplication of LETM1 generated an evolutionary conserved protein family ................................. 48 3.2. Letm1 and letm2 are ubiquitously expressed in larval and adult zebrafish tissues ....................................... 52 3.2.1 Zebrafish larvae display a ubiquitous letm1 and letm2 distribution ........................................................ 52 3.2.2 Letm1 and letm2 are ubiquitously expressed in the adult zebrafish brain .............................................. 53 3.2.3 Letm1 and letm2 are ubiquitously expressed in zebrafish and human cells ............................................ 54 3.3. Letm1 and Letm2 are highly conserved, mitochondrial associated proteins ................................................ 57 3.4. Establishment of a vital, haploinsufficient letm1 zebrafish line .................................................................... 59 3.5. Generation of a CRISPR/Cas9 mediated letm2 knock-out strain yield first viable larvae .............................. 60 3.6. Letm1 haploinsufficient mutants reveal an unchanged locomotor behavior ................................................ 61 4. Discussion ...................................................................................................................... 65 4.1. LETM1 and LETM2 are highly conserved proteins throughout the eukaryotic kingdom ............................... 65 4.2. The ubiquitously abundant LETM proteins may play a crucial role during development ............................. 66 4.3. Establishment of novel letm1 and letm2 zebrafish lines ............................................................................... 68 4.3.1 The generated letm1 haploinsufficient zebrafish strain will serve as an indispensable model to investigate several aspects of the mitochondrial protein .......................................................................