Dynamics of DNA methylation in differentiating hematopoietic cells [Elektronische Ressource] / vorgelegt von Maja Klug

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 18
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dynamics of DNA Methylation in Differentiating
Hematopoietic Cells


Dissertation zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen
Fakultät III - Biologie und vorklinische Medizin der Universität
Regensburg







vorgelegt von
Maja Klug aus Steinau
Juli 2009



The work presented in this thesis was carried out in the Department of Hematology and
Oncology at the University Hospital Regensburg from June 2005 to July 2009.


Die vorliegende Arbeit entstand in der Zeit von Juni 2005 bis Juli 2009 in der Abteilung für
Hämatologie und internistische Onkologie des Klinikums der Universität Regensburg.

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Promotionsgesuch eingereicht am: 20. Juli 2009
Die Arbeit wurde angeleitet von: PD Dr. Michael Rehli, Prof. Dr. Herbert Tschochner.



Prüfungsausschuss:
Vorsitzender: Prof. Dr. Armin Kurtz
1. Prüfer (Erstgutachten): Prof. Dr. Herbert Tschochner
2. Prüfer (Zweitgutachten): PD. Dr. Michael Rehli
3. Prüfer: Prof. Dr. Ernst Tamm

















Ever tried, ever failed, no matter,
Try again, fail again, fail better.
Samuel Beckett















Table of Contents
1 INTRODUCTION ............................................................................................. - 1 -
1.1 Epigenetics ............................................................................................................................ - 1 -
1.1.1 Molecular Building Blocks of Epigenetics........................................................................ - 1 -
1.1.1.1 Histone Modifications .................................................................................................. - 1 -
1.1.1.2 DNA Methylation.......................................................................................................... - 7 -
1.1.1.3 Non-Coding RNA.......................................................................................................- 11 -
1.2 Epigenetics in Hematopoiesis ........................................................................................... - 12 -
1.2.1 Hematopoiesis............................................................................................................... - 12 -
1.2.2 The Mononuclear Phagocyte System ........................................................................... - 13 -
1.2.2.1 Macrophages in the Immune Response.................................................................... - 13 -
1.2.2.2 Dendritic Cells in the Immune Response .................................................................. - 14 -
1.2.3 The Lymphoid Lineage..................................................................................................- 15 -
1.2.4 Role of Epigenetic Modifications for Lineage Commitment........................................... - 15 -
2 RESEARCH OBJECTIVES........................................................................... - 17 -
3 MATERIAL AND EQUIPMENT ..................................................................... - 18 -
3.1 Equipment............................................................................................................................ - 18 -
3.2 Consumables....................................................................................................................... - 19 -
3.3 Chemicals....... - 20 -
3.4 Enzymes and Kits................................................................................................................ - 20 -
3.5 Oligonucleotides.. - 21 -
3.5.1 cDNA Primer.................................................................................................................. - 21 -
3.5.2 ChIP/MCIP Primer.........................................................................................................- 22 -
3.5.3 Primer for in vivo Footprinting ....................................................................................... - 22 -
3.5.4 Primer for Cloning Experiments .................................................................................... - 23 -
3.5.5 Bisulfite Amplicon Generation (Nested PCR)................................................................ - 23 -
3.5.6 Bisution (MassARRAY)............................................................... - 23 -
3.6 Antibiotics............................................................................................................................ - 25 -
3.7 Plasmids............................................................................................................................... - 25 -
i 3.8 E.coli Strains........................................................................................................................ - 26 -
3.9 Antibodies............................................................................................................................ - 26 -
3.10 Cell Lines......... - 27 -
3.11 Databases and Software..................................................................................................... - 27 -
4 METHODS - 28 -
4.1 General Cell Culture Methods............................................................................................ - 28 -
4.1.1 Cell Line Culture............................................................................................................ - 28 -
4.1.1.1 Culture Conditions and Passaging............................................................................ - 28 -
4.1.1.2 Culturing of Stably Transfected Drosophila S2 Cells and Production of MBD-Fc..... - 29 -
4.1.1.3 Assessing Cell Number and Vitality .......................................................................... - 30 -
4.1.1.4 Freezing and Thawing Cells...................................................................................... - 30 -
4.1.1.5 Mycoplasma Assay.................................................................................................... - 30 -
4.1.2 Transient Transfection of Mammalian Cells.................................................................. - 31 -
4.1.2.1 Lipofectamine Transfection ....................................................................................... - 31 -
4.1.2.2 Transfection Using DEAE Dextran............................................................................ - 31 -
4.1.2.3 Measuring Luciferase Activity.................................................................................... - 32 -
4.1.3 Primary Cells ................................................................................................................. - 32 -
4.1.3.1 Isolation of Monocytes............................................................................................... - 32 -
4.1.3.2 Cultivation of Monocytes ........................................................................................... - 33 -
4.2 General Molecular Biology................................................................................................. - 34 -
4.2.1 Bacterial Culture............................................................................................................ - 34 -
4.2.1.1 Bacterial Growth Medium .......................................................................................... - 34 -
4.2.1.2 Transformation of Chemically Competent E.coli ....................................................... - 34 -
4.2.1.3 Glycerol Stock ...........................................................................................................- 35 -
4.2.2 Plasmid Isolation from E.coli ......................................................................................... - 35 -
4.2.3 Molecular Cloning.......................................................................................................... - 35 -
4.2.3.1 Construction of the pCpGL-basic Vector................................................................... - 35 -
4.2.3.2 Cloning of Reporter Vectors ...................................................................................... - 36 -
4.2.4 In Vitro Methylation of Plasmid DNA ............................................................................. - 37 -
4.2.5 Preparation and Analysis of DNA.................................................................................. - 37 -
4.2.5.1 Isolation and Quality Control of Genomic DNA ......................................................... - 37 -
4.2.5.2 Precipitation of DNA Using PEG (Polyethylene Glycol)............................................ - 37 -
4.2.5.3 Purification of DNA with Phenol Chloroform Extraction - 38 -
4.2.5.4 Agarose Gel Electrophoresis..................................................................................... - 38 -
4.2.5.5 Restriction Endonuclease Digestion..........................................................................- 39 -
ii 4.2.5.6 Dephosphorylation of DNA with Alkaline Phosphatase............................................. - 39 -
4.2.5.7 Fill in 5’-Overhangs with Klenow-DNA-Polymerase ..............................................