Aus der Klinik für Kardiologie und Pneumologie (Prof. Dr. med. G. Hasenfuß) der Medizinischen Fakultät der Universität Göttingen ‘Knockout-first’ mouse model as a biological tool to study the role of KIAA0182 gene in hypoplastic left heart syndrome INAUGURAL-DISSERTATION zur Erlangung des Doktorgrades der Medizinischen Fakultät der Georg-August-Universität zu Göttingen vorgelegt von Fouzi Alnour aus Damas Suburb, Syrien Göttingen 2015 Dekan: Prof. Dr. rer. nat. H. K. Kroemer I. Berichterstatter: Prof. Dr. E. Zeisberg II. Berichterstatter: Prof. Dr. S. Johnsen III. Berichterstatterin: Prof. Dr. M. Schӧn Tag der mündlichen Prüfung: 16.03.2016 Table of contents 1. Introduction .................................................................................................................. 1 1.1 Hypoplastic left heart syndrome .................................................................................... 2 1.1.1 Definition ....................................................................................................................... 2 1.1.2 Incidence ....................................................................................................................... 3 1.1.3 Pathogenesis and etiology ...................................................................................... 3 1.1.4 Genetics ........................................................................................................................ 5 1.1.5 Clinical presentation .................................................................................................. 6 1.1.6 Diagnosis and management .................................................................................... 6 1.2 Endocardial fibroelastosis (EFE) .................................................................................... 7 1.2.1 Definition ....................................................................................................................... 7 1.2.2 Classification ............................................................................................................... 7 1.2.3 Incidence ....................................................................................................................... 8 1.2.4 Etiology ......................................................................................................................... 8 1.2.5 Pathogenesis ............................................................................................................... 9 1.2.6 Diagnosis and management .................................................................................... 9 1.3 Endothelial to mesenchymal transition (EndMT) ..................................................... 10 1.3.1 Definition of EndMT ................................................................................................. 10 1.3.2 EndMT stimulants and mechanism ..................................................................... 10 1.3.3 EndMT markers ......................................................................................................... 12 1.3.4 EndMT and cardiac fibrosis ................................................................................... 13 1.4 KIAA0182 gene .................................................................................................................. 14 1.4.1 General information ................................................................................................. 14 1.4.2 Gse1 gene in mouse ................................................................................................ 15 1.4.3 KIAA0182 and circular RNA ................................................................................... 16 1.4.4 KIAA0182 and CoREST complex .......................................................................... 17 1.4.5 KIAA0182 and cardiovascular diseases ............................................................. 19 1.5 Gene trap mutagenesis ................................................................................................... 19 1.5.1 Mutagenesis strategies ........................................................................................... 19 1.5.2 Gene trapping ............................................................................................................ 20 1.5.3 The ‘Knockout-first’ strategy ................................................................................. 21 2. Materials and methods ................................................................................................23 2.1 Materials .............................................................................................................................. 23 2.1.1 Animals ....................................................................................................................... 23 2.1.2 Chemicals ................................................................................................................... 24 I 2.1.3 Commercial kits ........................................................................................................ 26 2.1.4 Cell culture mediums ............................................................................................... 26 2.1.5 Buffers ......................................................................................................................... 26 2.1.6 Instruments ................................................................................................................ 27 2.1.7 Antibodies .................................................................................................................. 28 2.1.8 Primers ........................................................................................................................ 28 2.1.9 Other materials .......................................................................................................... 31 2.2 Methods............................................................................................................................... 32 2.2.1 Genomic DNA extraction ........................................................................................ 32 2.2.2 RNA extraction .......................................................................................................... 32 2.2.3 RNA reverse transcription ...................................................................................... 33 2.2.4 RNase R treatment ................................................................................................... 33 2.2.5 DNA extraction from agarose gel ......................................................................... 34 2.2.6 Genotyping PCR ....................................................................................................... 34 2.2.7 Short-range PCR ....................................................................................................... 36 2.2.8 Long-range PCR........................................................................................................ 37 2.2.9 Reverse transcription PCR .................................................................................... 37 2.2.10 Quantitive real-time PCR ........................................................................................ 38 2.2.11 Isolation of mouse fibroblasts .............................................................................. 39 2.2.12 Cell culture ................................................................................................................. 39 2.2.13 EndMT assay ............................................................................................................. 40 2.2.14 Small interfering RNA transfection ...................................................................... 40 2.2.15 Transduction of primary fibroblasts with Cre-recombinase adenovirus ... 41 2.2.16 Protein extraction ..................................................................................................... 41 2.2.17 Western blotting........................................................................................................ 41 2.2.18 Ascending aortic constriction (AAC) .................................................................. 42 2.2.19 Masson's trichrome staining ................................................................................. 43 2.2.20 Statistical analysis ................................................................................................... 43 3. Results .........................................................................................................................45 3.1 Genotyping protocols for ‘Knockout-first’ mice ...................................................... 45 3.2 Quality control tests ........................................................................................................ 46 3.2.1 Confirming the specificity of Gse1 targeting ................................................... 46 3.2.2 Confirming the structure of the trapping cassette .......................................... 49 3.3 Genotyping results ........................................................................................................... 49 3.4 Generating mice with Gse1tm1c allele ........................................................................... 52 II 3.5 Generating Gse1tm1b allele in vitro .............................................................................. 54 3.6 Gse1 expression results ................................................................................................