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Gene Expression Profiling of Patients with Polycythemia Rubra Vera

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Gene Expression Profiling of Patients with Polycythemia Rubra Vera Gene Expression Profiling of Patients With Polycythemia Rubra Vera Generation of Transgenic Mice Expressing the Human PRV-1 Gene Inauguraldissertation Zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Diplom-Biochemiker Philipp Stefan Goerttler aus Freiburg im Breisgau Die vorliegende Arbeit wurde im Zeitraum von Juni 2000 bis August 2004 in der Klinik für Tumorbiologie und dem Zentrum für Klinische Forschung in der Abteilung für Experimentelle Anaesthesie der Albert-Ludwigs-Universität Freiburg unter der Betreuung von Frau Prof. Dr. Heike L. Pahl durchgeführt. Vor der Fakultät für Biologie wurde die Arbeit durch Herrn Prof. Dr. Gunther Neuhaus vertreten. Promotionsvortrag / Promotionsgespräch fanden am 26.10.2004 / 13.12.2004 statt. Prüfer: Prof. Dr. Heike L. Pahl (direkte Betreuung der Arbeit) Prof. Dr. G. Neuhaus (Vertretung der Arbeit vor der Fakultät für Biologie) Prof. Dr. C. Peters Priv. Doz. Dr. G. Scherer Prof. Dr. K. F. Fischbach (Prüfungs-Vorsitzender) Ich bestätige hiermit an Eides statt, daß die vorgelegte Arbeit von mir alleine und nur unter Zuhilfenahme der angegebenen Hilfsmittel durchgeführt wurde. Philipp Goerttler Freiburg, den 14.12.2004 1. Introduction........................................................................................................... 5 1.1 Chronic Myeloproliferative Disorders................................................................................ 6 1.2 Chronic Myeloid Leukaemia................................................................................................ 7 1.3 Essential Thrombocythaemia ............................................................................................. 9 1.4 Idiopathic Myelofibrosis.................................................................................................... 10 1.5 Polycythaemia Rubra Vera (PV) ....................................................................................... 11 1.5.1 Disease Pattern........................................................................................................... 11 1.5.2 Diagnosis..................................................................................................................... 12 1.5.3 Therapy ....................................................................................................................... 14 1.5.4 Molecular Characterisation of PV................................................................................ 15 1.5.5 Clonality.......................................................................................................................16 1.5.6 Progenitor Cell Assays ................................................................................................ 17 1.5.7 Growth Factor Sensitivity ............................................................................................ 18 1.5.8 Erythropoietin Receptor (EPO-R)................................................................................ 18 1.5.9 SHP-1 Phosphatase.................................................................................................... 19 1.5.10 STAT Family of Transcription Factors......................................................................... 19 1.5.11 Thrombopoietin Receptor............................................................................................ 20 1.5.12 Genomic Alterations in PV .......................................................................................... 21 1.5.13 Familial Polycythaemias.............................................................................................. 21 1.6 PRV-1................................................................................................................................... 22 1.6.1 Discovery and Characterisation of PRV-1................................................................... 22 1.6.2 Expression of PRV-1 mRNA in CMPDs ...................................................................... 23 1.6.3 Correlation Between PRV-1 Expression and EEC Growth ......................................... 23 1.6.4 Diagnostic Assay for the Determination of PRV-1 mRNA Levels ............................... 24 1.6.5 Expression of the PRV-1 Protein................................................................................. 24 1.6.6 A Murine Homologue of PRV-1 ................................................................................... 25 Aim of this work .............................................................................................................................. 26 2. Methods ............................................................................................................ 28 2.1 Whole Mount in-situ Hybridisation................................................................................... 28 2.1.1 Mating of Mice ............................................................................................................. 28 2.1.2 Killing of the Mice ........................................................................................................ 28 2.1.3 Preparation and Fixation of the Embryos.................................................................... 28 2.1.4 Dehydration ................................................................................................................. 28 2.1.5 Generation of DIG Labelled RNA Probes ................................................................... 28 2.1.6 In-Situ Hybridisation .................................................................................................... 29 2.2 DNA Preparation From Tail-biopsies of Mice.................................................................. 30 2.3 RNA Preparation ................................................................................................................ 31 1 2.3.1 RNA Isolation From Samples in TRIZOL®................................................................... 31 2.3.2 RNA Isolation From Granulocytes in GTC Solution .................................................... 31 2.3.3 Isolation of Total RNA From Mouse Embryos............................................................. 32 2.4 Quantification of Nucleic Acids........................................................................................ 32 2.5 Agarose Gel Electrophoresis............................................................................................ 32 2.5.1 RNA Electrophoresis ................................................................................................... 32 2.5.2 DNA Electrophoresis ................................................................................................... 33 2.5.3 Cleanup of DNA Fragments From Agarose Gels........................................................ 33 2.6 Northern Blot ...................................................................................................................... 33 2.6.1 Transfer of RNA to Nylon Membranes ........................................................................ 33 2.6.2 Generation of Radio-Labelled Probes......................................................................... 34 2.6.3 Membrane Hybridisation and Autoradiography........................................................... 34 2.7 Reverse Transcription (RT)............................................................................................... 34 2.8 Polymerase Chain Reaction (PCR)................................................................................... 35 2.8.1 Standard PCR Protocol ............................................................................................... 35 2.8.2 Colony PCR................................................................................................................. 36 2.8.3 Semi-Quantitative RT-PCR ......................................................................................... 36 2.9 FACS Analysis of Mouse Whole Blood............................................................................ 37 2.9.1 Single Colour Analysis (PRV-1) .................................................................................. 37 2.9.2 Multi Colour Analysis................................................................................................... 37 2.10 Sequence Analysis of DNA ............................................................................................... 38 2.11 Ligations ............................................................................................................................. 39 2.11.1 Ligation of Restriction Fragments Into Plasmid Vectors ............................................. 39 2.11.2 Ligation of PCR Products Using TOPOTM TA Cloning ................................................ 39 2.12 Transformation of Competent E. coli Cells ..................................................................... 39 2.13 Preparation of Plasmid DNA ............................................................................................. 40 2.14 TaqMan® Quantitative RT-PCR ......................................................................................... 40 2.14.1 Standard PRV-1 and GAPDH TaqMan® Assay........................................................... 42 2.14.2 Assays-on-DemandTM.................................................................................................. 43 2.15 Isolation of Granulocytes From Blood Samples............................................................. 44 2.16 Restriction Digest
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