Early target genes of CALM-AF10 as revealed by gene expression profiling [Elektronische Ressource] / submitted by Medhanie Assmelash Mulaw

Medhanie Assmelash Mulaw

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

From the Department of Medicine III, Grosshadern Hospital and
HelmholtzZentrum München, Clinical Cooperative Group “Leukemia”
Ludwig-Maximilians-University, Munich
Chair: Prof. Dr. med. Wolfgang Hiddemann






Early target genes of CALM/AF10 as revealed by
gene expression profiling





Thesis Submitted for a Doctoral degree in Human Biology
at the Faculty of Medicine Ludwig-Maximilians-University,
Munich, Germany




Submitted by
Medhanie Assmelash Mulaw



From
Asmara, Eritrea



2009
Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern
und HelmholtzZentrum München, Klinische Kooperations Gruppe ‚
“Leukämie”
der Ludwig-Maximilians-Universität München,
Direktor: Prof. Dr. med. Wolfgang Hiddemann






Frühe Zielgene von CALM/AF10 identifiziert
durch Genexpressionsprofilerstellung





Dissertation zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der Ludwig-Maximilians-
Universität zu München, Deutschland



vorgelegt von
Medhanie Assmelash Mulaw



Aus
Asmara, Eritrea


2009
Mit Genehmigung der Medizinischen Fakultät der
Universität München























Berichterstatter: Prof. Dr. med. Stefan K. Bohlander

Mitberichterstatter: Priv. Doz. Dr. Michael Albert
Prof. Dr. Wolfram Dempke

Mitbetreuung durch den
promovierten Mitarbeiter:
Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR, FRCR

Tag der mündlichen Prüfung: 27.07.2009














To my beloved wife, Blen Tsegaye Aregawi













Acknowledgement

I would like to extend my heartfelt gratitude to my supervisor Prof. Dr. med. Stefan K.
Bohlander for the opportunity and unreserved support throughout my study. His academic
and moral guidance was staggering and conspicuous, without which I couldn’t have managed
to complete this work in time.
I would also like to acknowledge all the members of the clinical cooperative group (CCG)
Leukemia, for the lively and vivid discussions that were quite informative and enlightening.
Former and current members of the Prof. Bohlander lab (Belay Tizazu, Deepak Bararia,
Alexandre Krause, Luciana-Fontanari Krause, Leticia Fröhlich Archangelo, Philipp Greif,
Zlatana Pašalić) are duly acknowledged, whose technical and collegial support was
instrumental in the completion of my study.
My deepest gratitude goes to my wife, Blen Tesgaye Aregawi, who has always supported my
dreams and encouraged me to fulfill them. Her love and support was pivotal for my study as
she patiently and understandingly stayed by my side during my PhD work, paying a gigantic
sacrifice that I am hugely indebted to.
I would also like to thank the German Academic Exchange Program (DAAD) for providing
me with the scholarship that made it easy for me to pursue my studies.
My acknowledgement also goes to my home institution, Addis Ababa University
(Department of Biology, Faculty of Science), which provided me with a study leave to carry
out my PhD work.
University of Munich and HelmholtzZentrum München are also duly acknowledged.
Contents

Acknowledgement .....................................................................................................................v
Contents ................................................................................................................................... vi
List of Tables .............................x
1.1. Genetic basis of cancer ....................................................................................................1
1.2. Chromosomal Translocations ..........................2
1.2.1. Definition ..................................................................................................................2
1.2.2. Types of chromosomal translocations ......2
1.2.3. Occurrence of chromosomal translocation ...............................................................3
1.2.4. Causes of chromosomal translocation ......................................3
1.2.5. Consequences of chromosomal translocations .........................................................4
1.3. Chromosomal translocations in hematopoietic malignancies .........................................5
1.4. CALM/AF10 .................................................................................10
II. Materials and Methods ........................................16
2.1. Materials ........................................................................................................................16
2.1.1. Chemicals ...............16
2.1.2. Enzymes .................................................................................................................21
2.1.3. Primers ....................23
2.1.4. Plasmids ..................................................................................................................23
2.1.5. Buffers and Solutions .............................24
2.1.6. Media ......................................................................................................................29
2.1.7. Antibodies...............30
2.1.8. Cells ........................................................................................................................30
2.1.9. Computer Operating System and Application Software ........................................31
2.1.10. Equipments and Utensils ......................................................................................32
2.2. Methods .........................................................................................................................34
2.2.1. Extraction ...............34
2.2.2. DNA digestions and modifications ........................................................................40
2.2.3. Transformation .......................................45
2.2.4. Electroporation .......................................................................46
2.2.5. Electrophoresis .......................................46
2.2.5. Gel Purification ......................................................................47
2.2.6. Cell Culture ............................................48
2.2.7. Transfection ............................................................................53
2.2.8. Electroporation .......................................53
2.2.9. Polymerase Chain Reaction (PCR) ........................................................................55
2.2.10. Colony PCR ..........................................60
2.2.11. Real Time PCR (Applied Biosystems 7900HT Micro Fluidic Card)...................61
2.2.12. Microarray Analysis .............................................................................................64
III. Results ................................................................70
3.1. Cloning experiments .....................................................................70
3.1.1. Cloning of FLAG-CALM/AF10 into the pUC19 Shuttle Vector ..........................70
3.1.2. Cloning of VP16 upstream of FLAG-CALM/AF10 ..............................................70
3.1.3. Cloning of FLAG-CALM/AF10 and FLAG-VP16-CALM/AF10 into pRTS-1 ....73
3.2. Establishing a stably transfected cell line .....................................................................74
3.2.1. Electroporation/transfection ...................................................................................75
3.2.2. Selection/enrichment of transfected cells ...............................76
3.2.3. Conditional expression ...........................................................................................77
3.3. RT PCR .........................................................79
3.4. Microarray .....................................................................................81 3.4.1. Experimental Setup ................................................................................................81
3.4.2. Low-level Analysis of Microarray Data .81
3.4.3. High-level analysis .................................................................................................86
3.4.4. Chromosomal distribution of differentially regulated genes ..99
3.4.5. Ontology Analysis (GOSurfer).............................................................................104
3.4.6. Pathway Analysis (KegArray) ..............109
3.4.7. Comparison with patient data (Gene Set Enrichment Analysis, GSEA) ..............114
® 3.5. Verification of Potential Target Genes by Real Time PCR (Taqman Low Density
Array) .................................................................................................................................117
3.5.1. Comparison between array and LDA data ........