Development of novel RNA and protein based cancer therapeutics by either silencing of potential oncogenes or active restoration of a tumor suppressor gene [Elektronische Ressource] / vorgelegt von Inga Neef

Tom Leahy

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Development of novel RNA- and protein-based cancer
therapeutics by either silencing of potential oncogenes
or active restoration of a tumor suppressor gene

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der
Rheinisch- Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigte Dissertation
vorgelegt von

Diplom Bioinformatikerin
Inga Neef
aus Bingen am Rhein


Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Dr. rer. medic. Stefan Barth

Tag der mündlichen Prüfung: 16.03.2009

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
Index 2
1 Introduction.............................................................................................................6
1.1 General background ......................................................................................6
1.2 Cancer...........................................................................................................8
1.3 Molecular carcinogenesis ..............................................................................9
1.4 Targeted cancer therapy..............................................................................12
1.5 The lymphocyte activation marker CD30 .....................................................14
1.6 Human death associated protein kinase ......................................................15
1.7 Novel strategies for targeted cancer therapy................................................17
1.8 RNA interference .........................................................................................17
1.9 RNA interference for cancer therapy............................................................22
1.10 Aptamers .....................................................................................................22
1.11 Novel target gens for RNA interference .......................................................24
1.12 Selective systemic delivery of siRNAs .........................................................25
1.13 Prostate specific membrane antigen (PSMA)...............................................29
1.14 Human epidermal growth factor receptor 3: ERbB3/HER 3..........................29
1.15 Aim of the thesis ..........................................................................................30
2 Materials and Methods..........................................................................................35
2.1 Materials......................................................................................................35
2.1.1 Chemicals ............................................................................................35
2.1.2 Media stock solutions and buffers ........................................................35
2.1.3 Standard buffer and media compositions .............................................35
2.1.4 Buffers for RNA work ...........................................................................37
2.1.5 Buffers for protein work ........................................................................38
2.1.6 Antibodies ............................................................................................39
2.1.7 Reaction kits and enzymes ..................................................................39
2.1.8 Bacterial strains and vector systems....................................................40
2.1.9 Equipment and applications .................................................................41
2.2 Methods.......................................................................................................42
2.2.1 Cell culture...........................................................................................42
2.2.1.1 For Immuno-RNA-transcripts........................................................42
2.2.1.2 For Immunokinases......................................................................42
2.2.2 Electrophoresis protocols.....................................................................42
2.2.2.1 Agarose gel electrophoresis .........................................................42
2.2.2.2 Analytical SDS PAGE...................................................................42
2.2.2.3 Urea PAGE ..................................................................................43
2.2.2.4 Analytical native PAGE.................................................................43
2.2.3 Methods for the evaluation of siRNA-mediated gene silencing.............43
2.2.3.1 siRNA transfection........................................................................43
2.2.3.2 Analysis of transfection efficiencies by fluorescence microscopy..43
2.2.3.3 Total RNA preparation..................................................................43
2.2.3.4 First strand cDNA synthesis .........................................................44
2.2.3.5 siRNA deprotection and annealing ...............................................44
2.2.3.6 Quantitative real time RT-PCR analysis (qPCR)...........................44 Index 3
2.2.3.7 Western blot analysis for protein silencing....................................44
2.2.3.8 Cell viability assay........................................................................45
2.2.4 Methods for the preparation of RNA aptamers .....................................45
2.2.4.1 RNA secondary structure prediction .............................................45
2.2.4.2 Assembly polymerase chain reaction ...........................................45
2.2.4.3 Polymerase chain reaction ...........................................................46
2.2.4.4 DNA sequence analysis ...............................................................46
2.2.4.5 Phenol chloroform extraction........................................................47
2.2.4.6 Ethanol precipitation.....................................................................47
2.2.4.7 in vitro transcription ......................................................................47
2.2.4.8 Measurement of DNA and RNA concentrations............................47
2.2.4.9 Fluorescence labeling of RNA ......................................................48
2.2.5 Aptamer-siRNA transcript evaluation ...................................................48
2.2.5.1 Flow cytometric binding analysis ..................................................48
2.2.5.2 Cell-surface affinity measurement of aptamer-siRNA transcripts..48
2.2.5.3 Internalization assay.....................................................................49
2.2.5.4 Interferon assay........................................................................49
2.2.5.5 DICER cleavage assay.................................................................50
2.2.5.6 Apoptosis assay ...........................................................................50
2.3 Methods for immunokinases ........................................................................50
2.3.1 Construction of Immunokinases ...........................................................50
2.3.1.1 Bacterial strains and oligonucleotides...........................................50
2.3.1.2 Polymerase chain reaction ...........................................................50
2.3.1.3 Cloning.........................................................................................51
2.3.1.4 Eukaryotic cell transfection and recombinant protein production ..51
2.3.1.5 Nucleofection of eukaryotic cells ..................................................52
2.3.1.6 Protein purification........................................................................52
2.3.1.7 SDS-PAGE and Western Blot analysis (2.2.2.2, 2.2.3.7)..............52
2.3.1.8 Mass spectrometry.......................................................................52
2.3.2 Methods for screening different cell lines for DAPK2 expression..........53
2.3.2.1 Western blot analysis of cell lysates .............................................53
2.3.2.2 DNA isolation, bisulfite treatment and methylation-specific PCR ..53
2.3.2.3 RNA isolation and reverse transcription polymerase chain reaction
(RT-PCR) .....................................................................................53
2.3.2.4 Drug treatment .............................................................................54
2.3.3 Methods for Immunokinase evaluation.................................................54
2.3.3.1 in vitro kinase assay.....................................................................54
2.3.3.2 Flow cytometric binding assay......................................................54
2.3.3.3 Cell-surface affinity measurements...............................................55
2.3.3.4 Proliferation assay (2.2.3.8)..........................................................55
2.3.3.5 Apoptosis assay ...........................................................................55
2.3.3.6 in vitro mouse serum stability .......................................................56
2.3.4 Animal experiments.............................................................................56 Index 4
2.3.4.1 in vivo SCID mouse studies...........................................