Analysis of the leukemogenic potential of the CALM-AF10 fusion gene in patients, transgenic mice and cell culture models [Elektronische Ressource] / by Alexandre Krause

Alexandre Krause

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

From the Department of Internal Medicine III, Ludwig Maximilian University Munich
Director: Univ.-Prof. Dr. med. W. Hiddemann

Work performed under direction of Prof. Dr. med. Stefan K. Bohlander
Department of Internal Medicine III, Ludwig-Maximilian-University Munich,
GSF- Clinic Cooperative Group “Leukemia”.

Presented by Prof. Dr. med. vet. Eckhard Wolf
Institute of Molecular Animal Breeding and Biotechnology, Gene Center
Faculty of Veterinary Medicine of the Ludwig-Maximilians-University Munich








Analysis of the Leukemogenic Potential of the CALM/AF10 Fusion Gene in Patients,
Transgenic Mice and Cell Culture Models






Thesis for the attainment of the title Doctor in Veterinary Medicine from the Faculty of
Veterinary Medicine of the Ludwig-Maximilians-University, Munich




By
Alexandre Krause
From São Miguel do Oeste
Munich 2006


Aus der Medizinischen Klinik und Poliklinik III,
Klinikum der Ludwig Maximilians Universität München, Großhadern
Direktor: Univ.-Prof. Dr. med. W. Hiddemann

Arbeit unter der Leitung von
Prof. Dr. med. Stefan K. Bohlander
Klinikum der Ludwig Maximilians Universität München, Großhadern,
GSF, Klinische Kooperationsgruppe „Pathogenese der akuten myeloischen Leukämie“

Vorgelegt über Prof. Dr. med. vet. Eckhard Wolf
Institut für molekulare Tierzucht
der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München







Untersuchung des leukämogenen Potentials des CALM-AF10 Fusionsgens im
Patienten, im transgenen Mausmodell und in Zellkulturmodellen






Inaugural-Dissertation
Zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München




Von
Alexandre Krause
Aus São Miguel do Oeste
München 2006


Gedruckt mit Genehmigung der Tierärzlichen Fakultät der
Ludwig-Maximilians-Universität München









Dekan: Univ.-Prof. Dr. E. P. Märtlbauer

Referent: Univ.-Prof. Dr. E. Wolf

Korreferent: Univ.-Prof. Dr. H.-J. Gabius






Tag der Promotion: 10. Februar 2006





















To my Family



Table of contents i

TABLE OF CONTENTS

1. ABBREVIATIONS ________________________________________________________ 1
2. INTRODUCTION AND OBJECTIVES _______________________________________ 4
3. REVIEW OF THE LITERATURE___________ 5
3.1. Hematopoiesis and Stem Cells _________________________________________________ 5
3.1.1. Early Hematopoiesis ______________________________________________________________ 5
3.1.2. Definitive Hematopoiesis __________________________________________________________ 5
3.1.3. Stem cells_______________________________________________________________________ 6
3.2. Leukemia and Leukemic Stem Cells ___________________________________________ 10
3.3. Chromosomal Translocations_________________________________________________ 11
3.4. The t(10;11)(p13;q14) _______________________________________________________ 12
3.5. CALM, AF10 and CALM/AF10_______________________________________________ 13
3.5.1. CALM_________________________________________________________________________ 13
3.5.2. AF10 _________________________________________________________________________ 15
3.5.3. CALM/AF10____________________________________________________________________ 18
3.6. HOX genes ________________________________________________________________ 19
3.7. The transgenic mouse model _________________________________________________ 22
3.7.1. Transgenic animals ______________________________________________________________ 22
3.7.2. Tissue-specific gene expression_____________________________________________________ 23
3.8. Inducible gene expression ____________________________________________________ 24
4. MATERIALS AND METHODS_____________________________________________ 27
4.1 Patients ___________________________________________________________________ 27
4.2 Animals ___________________________________________________________________ 27
Breeding strategy __________________________________________________________________ 27
4.3 Material and Reagents _______________________________________________________ 28
4.4 Media and Solutions _________________________________________________________ 34
4.4.1Cell Culture _____________________________________________________________________ 34
4.4.2 Western blotting _________________________________________________________________ 34
4.4.3 Southern Blotting ________________________________________________________________ 36
4.4.4 Bacterial Culture, DNA preparation/manipulation/extraction ______________________________ 37
4.4.5 FACS Solutions _________________________________________________________________ 38
4.5 Methods ___________________________________________________________________ 39
4.5.1 Chemically Competent Bacteria _____________________________________________________ 39
4.5.2 Electrocompetent Bacteria _________________________________________________________ 39
4.5.3 Bacterial Transformation __________________________________________________________ 39
Chemical Transformation ____________________________________________________________ 39
Electroporarion ____________________________________________________________________ 40
4.5.4 Plasmid Preparation ______________________________________________________________ 40
Miniprep _________________________________________________________________________ 40
4.5.5 Determination of DNA concentration_________________________________________________ 41
4.5.6 Agarose Gel Electrophoresis _______________________________________________________ 41
4.5.7 DNA Gel Extraction ______________________________________________________________ 41
4.5.8 Restriction enzyme digestion of plasmid and genomic DNA _______________________________ 42
4.5.9 DNA cloning____________________________________________________________________ 42
4.5.10 Automated fluorescent sequencing of PCR products or plasmid DNA ______________________ 43
4.5.11 Cell culture ____________________________________________________________________ 43
4.5.12 Examination of CALM/AF10 transgenicity in FVB mice by PCR and Southern blotting ________ 44
Table of contents ii

Mouse-tail genomic DNA extraction ___________________________________________________ 44
Polymerase Chain Reaction (PCR) _____________________________________________________ 44
Southern Blotting __________________________________________________________________ 45
DNA labelling_____________________________________________________________________ 46
DNA hybridization on nitrocellulose membranes__________________________________________ 46
Film Exposure_____________________________________________________________________ 47
4.5.13 Transient transfection of mammalian cells ____________________________________________ 47
Cell fixation for microscopy __________________________________________________________ 48
4.5.14 Stable transfection of mammalian cells ______________________________________________ 48
4.5.15 Obtaining and processing of microscope images _______________________________________ 48
4.5.16 Examination of CALM/AF10 expression at RNA Level by Reverse Transcriptase PCR and
Microarrays _________________________________________________________________________ 48
RNA extraction from frozen cells______________________________________________________ 48
RNA from FACS-sorted cells_________________________________________________________ 50
RNA preparation from mouse tissue____________________________________________________ 50
cDNA synthesis (Reverse Transcriptase Reaction) ________________________________________ 50
4.5.17 Western blotting ________________________________________________________________ 51
Sample preparation _________________________________________________________________ 51
Determination of Protein Concentration _________________________________________________ 51
Protein gel electrophoresis ___________________________________________________________ 52
Western blotting and Detection________________________________________________________ 52
4.5.18 Blood analysis of transgenic mice __________________________________________________ 53
4.5.19 Flow cytometry_________________________________________________________________ 54
4.5.20 Post mortem analysis of transgenic mice _____________________________________________ 54
5. RESULTS ______________________________________________________________ 55
5.1. Cell Biology _______________________________________________________________ 55
5.1.1.Sequencing of CALM/AF10________________________________________________________ 55
5.1.2.Sub-cellular loc