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'Knockout-First' Mouse Model As a Biological Tool to Study the Role of KIAA0182 Gene in Hypoplastic Left Heart Syndrome

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'Knockout-First' Mouse Model As a Biological Tool to Study the Role of KIAA0182 Gene in Hypoplastic Left Heart Syndrome 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 ................................................................................................
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