Expression and functional analysis of EFNB1 mutations in craniofrontonasal syndrome [Elektronische Ressource] / von Roman Makarov

Expression and functional analysis of EFNB1 mutations in craniofrontonasal syndrome Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität Magdeburg von Diplom-Genetiker Roman Makarov geboren am 9.07.1983 in Pushchino, Russland Gutachter: PD Dr. Ilse Wieland Prof. Dr. Ingo Hansmann eingereicht am: 26.10.2009 verteidigt am: 26.05.2010 Contents 1. Introduction 41.1. Ephrin binding receptors (Eph) 4 1.2 Ephrins 51.3. Eph–ephrin signalling system 7 1.4. Ephrin-B1 and EphB2 8 1.5. Ephrin – Eph signalling in vascular and nervous systems 15 1.6. EFNB1 gene and CFNS 18 1.7. Hypothesis ofcellular interference 21 1.8. The aims ofthe work 23 2. Materials and methods 25 2.1 Cels cultres work 5 2.1.1. Cell cultures maintaining 25 2.1.2. NIH 3T3 transfection 5 2.1.3. FACS analysis 6 2.1.4. NIH 3T3 stimulation 6 2.1.5. Patches formation analysis 27 2. Molecular methods 27 2.2.1. DNA extraction from fibroblasts 27 2.2. Mutaion detcion 8 2.2.3. RNA extraction with NucleoSpin® RNA / Protein 29 2.2.4. RT-PCR and cloning 30 2.2.5. Real-time RT-PCR 32 2.2.6. Generation of the mutant EFNB1 cDNA constructs by site-directed in vitro mutagenesis 33 2.7. Sequencig 34 2.2.8. Western blot analysis 6 3. Results 8 3.1.