Gene expression profiling of patients with polycythemia rubra vera [Elektronische Ressource] : generation of transgenic mice expressing the human PRV-1 gene / vorgelegt von Philipp Stefan Goerttler

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• Gesundheit

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

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 eloid 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 Elecis 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
TM2.11.2 Ligation of PCR Products Using TOPO 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
TM2.14.2 Assays-on-Demand .................................................................................................. 43
2.15 Isolation of Granulocytes From Blood Samples