Studying the role of the fusion protein MLL-AF4 in leukemogenesis with the help of siRNAs [Elektronische Ressource] / von Maria Thomas (geb. Arkhipova)

Tomik

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Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2007
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Studying the role of the fusion protein MLL-AF4 in
leukemogenesis with the help of siRNAs



der Fakultät für Biologie
der Eberhard Karls Universität Tübingen


zur Erlangung des Grades eines Doktors
der Naturwissenschaften


von
Maria Thomas
(geb. Arkhipova)
aus Pushchino (Russland)
vorgelegte

D i s s e r t a t i o n



2007






















Tag der mündlichen Prüfung: 11.10.2006
Dekan: Professor Dr. F. Schöffl
1. Berichterstatter: Professor Dr. A. Nordheim
2. Berichterstatter: PD Dr. O. Heidenreich
3. Berichterstatter: Professor Dr. R. Marschalek







To my deeply beloved parents,
my first teachers and scientific encourages,
my strictest judges and closest friends
PUBLICATIONS




PUBLICATIONS

During the course of this work the following articles have been published
or submitted for publication:

Thomas M, Gessner A, Vornlocher HP, Hadwiger P, Greil J,
Heidenreich O. (2005). Targeting MLL-AF4 with short interfering RNAs
inhibits clonogenicity and engraftment of t(4;11)-positive human leukemic cells.
Blood. 106, 3559-66.

Thomas M, Greil J, Heidenreich O. (2006). Targeting leukemic fusion
proteins with small interfering RNAs: recent advances and therapeutic
potentials. Acta Pharmacol Sinica. 27, 273-81.

Thomas M, Martinez Soria N, Heidenreich O. (2006). RNA Interference
in Hematopoietic and Leukemic Cells. EDS. Systems of Biology, pp. 29-58,
Springer Publishing House.TABLE OF CONTENTS
SUMMARY.................................................................................................................. i
ZUSAMMENFASSUNG ............................................................................................. ii
I. INTRODUCTION ..................................................................................................... 1
I.1Hallmarks of ALL ............................................................................................................................ 2
I.2 General aspects and molecular characteristics of infant acute lymphoblastic leukemia .............. 4
I.2.1 Diversity of translocation partners - common themes........................................................................... 5
I.2.2 MLL fusions and HOX gene expression ............................................................................................... 7
I.2.3 Other characteristic features of MLL-associated leukemia................................................................... 7
I.3 MLL protein.................................................................................................................................... 9
I.3.1 MLL and its fusions have an epigenetic function................................................................................ 11
I.4 t(4;11) translocation..................................................................................................................... 13
I.4.1 MLL-AF4 and AF4-MLL...................................................................................................................... 13
I.5 RNA interference......................................................................................................................... 15
I.5.1 Short overview of the siRNA mechanism ........................................................................................... 16
I.5.2 siRNA design and delivery ex vivo ..................................................................................................... 18
I.6 Aim of the project ........................................................................................................................ 20
II. MATERIALS......................................................................................................... 21
II.1 Devices....................................................................................................................................... 21
II.2 Buffers and solutions.................................................................................................................. 21
Cell culture ................................................................................................................................................. 21
Protein extracts and western blotting ......................................................................................................... 22
E.coli transformation................................................................................................................................... 24
Hybridization of siRNAs.............................................................................................................................. 24
Reverse Transcription and real-time PCR.................................................................................................. 25
Colony formation assay.............................................................................................................................. 25
Cell cycle analysis ...................................................................................................................................... 26
Telomerase activity assay .......................................................................................................................... 26
ChIP assay................................................................................................................................................. 27
II.3 Kits.............................................................................................................................................. 28
II.4 Enzymes..................................................................................................................................... 28
II.5 Antibodies................................................................................................................................... 29
II.6 Fluorescent dyes ........................................................................................................................ 31
II.7 Synthetic oligonucleotides.......................................................................................................... 31
siRNAs ....................................................................................................................................................... 31
Primers for conventional PCR .................................................................................................................... 33
Primers for methylation-specific PCR......................................................................................................... 33
Primers for quantitative real-time RT-PCR ................................................................................................. 34
II.8 Plasmids..................................................................................................................................... 35
II.9 Cell lines..................................................................................................................................... 35
III. METHODS........................................................................................................... 37
III.1 Abbreviations............................................................................................................................. 37
III.1. Cell culture................................................................................................................................ 38
Cell lines..................................................................................................................................................... 38
Freezing and thawing of the cell lines ........................................................................................................ 38
III.2 Transfections............................................................................................................................. 39
Electroporation ........................................................................................................................................... 39
Nucleophection........................................................................................................................................... 39
III.3 RNA isolation............................................................................................................................. 39
III.4 Protein lysate preparation ......................................................................................................... 40
III.5 Real-time RT-PCR .................................................................................................................... 40
cDNA synthesis .......................................................................................................................................... 40
Taqman PCR.............................................................................................................................................. 41
III.6 Colony formation assay...............................................................................................