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Characterization of Ceramide Synthase 2 Dysfunctions Caused by Targeted Mutations in Transgenic Mice

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Characterization of Ceramide Synthase 2 Dysfunctions Caused by Targeted Mutations in Transgenic Mice Characterization of Ceramide synthase 2 dysfunctions caused by targeted mutations in transgenic mice - Dissertation - zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Martina van Uelft (geb. Klahre) aus Elsterwerda Bonn, 2019 Angefertigt mit Genehmigung der Mathematisch- Naturwissenschaftlichen- Fakultät der Rheinischen- Friedrich- Wilhelms- Universität Bonn. Erstgutachter: PD Dr. Reinhard Bauer Zweitgutachter: Prof. Dr. Walter Witke Tag der Promotion: 10.01.2020 Erscheinungsjahr: 2020 Eidesstattliche Erklärung Eidesstattliche Erklärung Hiermit versichere ich, dass diese Dissertation von mir selbst und ohne Hilfe angefertigt worden ist. Es wurden keine anderen als die angegebenen Hilfsmittel benutzt. Ferner erkläre ich, dass die vorliegende Arbeit an keiner anderen Universität als Dissertation eingereicht wurde. Teile dieser Arbeit wurden bereits in folgender Originalpublikation veröffentlicht: Bickert A, Kern P, van Uelft M, Herresthal S, Ulas T, Gutbrod K, Breiden B, Degen J, Sandhoff K, Schultze JL, Dörmann P, Hartmann D, Bauer R, Willecke K. Inactivation of ceramide synthase 2 catalytic activity in mice affects transcription of genes involved in lipid metabolism and cell division. Biochim Biophys Acta Mol Cell Biol Lipids. 2018 Jul; 1863(7):734-749. Bonn, MARTINA VAN UELFT 1 Content Content 1 Introduction ................................................................................................................................... 1 1.1 Membranes............................................................................................................................. 1 1.2 Lipids ........................................................................................................................................ 2 1.3 Sphingolipids......................................................................................................................... 2 1.3.1 Ceramide ........................................................................................................................ 3 1.3.2 Sphingosine-1-phosphate and Ceramide-1-phosphate ............................... 5 1.4 Ceramide synthases ............................................................................................................ 6 1.4.1 Protein domains and motifs .................................................................................... 6 1.4.2 Expression pattern and substrate specificity ..................................................... 8 1.4.3 CerS1 and CerS1-deficient mice ............................................................................ 8 1.4.4 CerS2 and CerS2- deficient mice ........................................................................... 8 1.4.5 CerS3 and CerS3- deficient mice ........................................................................... 8 1.4.6 CerS4 and CerS4- deficient mice ........................................................................... 9 1.4.7 CerS5 and CerS5- deficient mice ........................................................................... 9 1.4.8 CerS6 and CerS6- deficient mice ........................................................................... 9 1.5 Drosophila melanogaster ceramide synthase - schlank ........................................ 9 1.6 Ceramides and energy metabolism ............................................................................ 10 1.6.1 Ceramide and sphingolipids in the development of metabolic disorders 10 1.7 Immune system .................................................................................................................. 12 1.7.1 Thymus .......................................................................................................................... 12 1.7.2 T cell development - Thymopoiesis .................................................................... 13 1.7.3 S1P signalling and thymic egress ........................................................................ 14 1.8 Manipulation of gene function ..................................................................................... 15 1.8.1 Conventional gene targeting via homologous recombination ................ 15 2 Aim of the study ........................................................................................................................ 17 3 Material ......................................................................................................................................... 18 3.1 Buffer, stock solutions and cell culture media ........................................................ 18 3.1.1 Nucleic acid sample preparation buffer and stock solutions .................... 18 3.1.2 Protein preparation buffer; and stock solutions ............................................ 19 3.1.3 Bacterial cell culture; buffer and stock solutions ........................................... 20 3.1.4 Embryonic stem cell culture; medium, buffer and stock solutions ......... 21 3.1.5 Cell culture; medium, buffer and stock solutions .......................................... 22 3.1.6 FACS experiments; buffer and stock solutions ............................................... 22 3.1.7 Lipid Biochemistry ..................................................................................................... 23 3.1.8 Histological staining solutions ............................................................................. 23 3.2 Antibodies ............................................................................................................................ 24 3.2.1 Primary antibodies .................................................................................................... 24 3.2.2 Secondary antibodies .............................................................................................. 24 3.2.3 FACS antibodies ......................................................................................................... 24 3.3 Primer molecules ............................................................................................................... 25 3.4 Plasmids ................................................................................................................................ 26 3.5 Hybridization probes ........................................................................................................ 26 3.6 Bacterial artificial chromosomes .................................................................................. 26 3.7 Eukaryotic cell lines ........................................................................................................... 26 3.8 Bacterial strains ................................................................................................................... 27 3.9 Transgenic mouse lines ................................................................................................... 27 II Content 3.10 DNA ladder and Protein marker............................................................................... 28 3.11 Lipid Standards for TLC Analyses ............................................................................. 28 4 Methods ........................................................................................................................................ 29 4.1 Nucleic acid biochemistry............................................................................................... 29 4.1.1 Analytical plasmid preparation (Mini prep, quick & dirty) ......................... 29 4.1.2 Preparative plasmid isolation (Midi prep) ........................................................ 29 4.1.3 Endotoxin-free preparation (Maxi prep) ........................................................... 29 4.1.4 Isolation of genomic DNA ...................................................................................... 29 4.1.5 Photometric estimation of DNA/RNA concentration ................................... 30 4.1.6 Sequence validation ................................................................................................. 30 4.2 Molecular cloning .............................................................................................................. 30 4.2.1 Restriction digestion, dephosphorylation and ligation ............................... 30 4.2.2 Generation of competent cells, transformation and “Recombineering” .... .......................................................................................................................................... 31 4.3 Eukaryotic cell culture ...................................................................................................... 33 4.3.1 Cell counting and calculations (Neubauer chamber) ................................... 33 4.3.2 HEK cell culture .......................................................................................................... 33 4.3.3 MEF cell culture .......................................................................................................... 33 4.3.4 Transient transfection with Metafectene (S10 cell culture dish) .............. 33 4.3.5 HM1 embryonic stem cell culture (mouse) ...................................................... 34 4.4 Generation, handling and analysis of transgenic mice ........................................ 35 4.4.1 Generation of the CerS2Flox, CerS2H212A/H213A & CerS2Del79-120 vector ...... 35 4.4.2 Generation of the conditional CerS2Del 79-120 vector .............................. 37 4.4.3
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