Genetic modification of oncolytic adenoviruses for anti-cancer-therapy [Elektronische Ressource] / presented by Christina Quirin

Dani Plana Trenchs And Mara Wish

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences

















Presented by

Diplom Biologin Christina Quirin
born in: Munich
Oral-examination:










Genetic Modification of Oncolytic Adenoviruses
for Anti-Cancer-Therapy


















Referees: PD Dr. Suat Özbek
PD Dr. Dirk NettelbeckTable of Contents
Zusammenfassung ...................................................................1 
1.  Summary ..............................................................................2 
2.  Introduction .........................................................................3 
2.1.  Cancer and cancer therapies ............................................................... 3 
2.2.  Gene therapy for cancer treatment ...................................................... 4 
2.3.  Virotherapy for cancer treatment ......................................................... 8 
2.4.  Adenovirus and their use as gene therapy vector or oncolytic virus
................................................................................................................ 9 
2.4.1.  Adenoviruses: Virion Structure, Cell Entry and Genome Organization ......... 10 
2.4.1.1.  Serotypes and Virus Structure .................................................................................... 10 
2.4.1.2.  Cell Binding and Entry ................................................................................................. 11 
2.4.1.3.  Genome Organization and Viral Replication ............................................................... 12 
2.4.2.  Adenoviral vectors15 
2.4.3.  Oncolytic adenoviruses ......................................................................................... 16 
2.4.3.1.  Strategies for restricting Ad replication to tumour cells ............................................... 17 
2.4.3.2. s for tropism modification of adenoviruses ................................................... 19 
2.4.3.3.  Strategy for efficient transgene expression by oncolytic adenoviruses ...................... 22 
3.  Objectives of the study ................................................... 24 
4.  Materials and Methods .................................................... 25 
4.1.  Materials ............................................................................................... 25 
4.1.1.  Chemicals, filters and enzymes ........................................................................... 25 
4.1.2.  Buffers and solutions ............................................................................................. 25 
4.1.2.1.  Buffers and solutions for gel electrophoresis .............................................................. 25 
4.1.2.1.1.  Electrophoresis of nucleic acids ............................................................................. 25 
4.1.2.1.2. phoresis of proteins .................................................................................... 25 
4.1.2.2.  Buffers and solutions for western blot analysis ........................................................... 26 
4.1.2.3.  Buffers and solutions for viral lysis .............................................................................. 26 
4.1.2.4.  Buffers and solutions for production of transformation competent bacteria ................ 26 
4.1.2.5.  Buffers ans for DNA precipitation ................................................................ 26 
4.1.2.6. d solutions for caesium chloride equilibrium density ultracentrifugation ..... 26 
4.1.3.  Media ....................................................................................................................... 26 
4.1.3.1.  Media for bacterial culture ........................................................................................... 26 
4.1.3.2.  Media and solutions for cell culture ............................................................................. 27 
4.1.4.  Cells and Bacteria Strains27 
4.1.4.1.  Bacteria strains ............................................................................................................ 27 
4.1.4.2.  Human cells lines ........................................................................................................ 28 
4.1.5.  Adenoviruses .......................................................................................................... 28 
4.1.6.  Nucleic acids ........................................................................................................... 29 
4.1.6.1.  Oligonucleotides29 
4.1.6.1.1.  Oligonucleotides for PCR cloning .......................................................................... 29 
4.1.6.1.2. ucleotides for annealing ............................................................................... 29 
4.1.6.1.3.  Oligonuclsplicing analysis .................................................................... 29 
4.1.6.1.4.  Oligonuclcontrolling recombinant modified Ad genomes ..................... 30 
4.1.6.1.5. ucleotides for sequencing ............................................................................ 31 Table of Contents
4.1.6.1.6.  Oligonucleotides for quantitative real time PCR (qPCR) ....................................... 31 
4.1.6.2.  Plasmids ...................................................................................................................... 32 
4.1.6.3.  Antibodies .................................................................................................................... 33 
4.1.6.3.1. s for western blot analysis ....................................................................... 33 
4.2.  Methods ................................................................................................ 34 
4.2.1.  Nucleic acid methods ............................................................................................ 34 
4.2.1.1.  DNA cloning ................................................................................................................. 34 
4.2.1.1.1.  Production of transformation-competent bacteria and transformation ................... 34 
4.2.1.1.1.1.  Production of chemical-competent bacteria and transformation by heat shock
...................................................................................................................... 34 
4.2.1.1.1.2.  Production of electro-competent bacteria and tran
electroporation .............................................................................................. 35 
4.2.1.1.1.3.  Homologous recombination for the generation of recombinant adenoviral
genomes ....................................................................................................... 35 
4.2.1.2.  Preparation of DNA and RNA ...................................................................................... 36 
4.2.1.2.1.  Analytical isolation of plasmid DNA (mini lysate) ................................................... 36 
4.2.1.2.2.  Quantitative isolation of plasmid DNA (midi lysate) ............................................... 36 
4.2.1.2.3.  DNA isolation from infected human cell cultures37 
4.2.1.2.4.  RNA isolation ......................................................................................................... 37 
4.2.1.2.5.  and reverse transcription................................................................. 37 
4.2.1.3.  PCR (poymerase chain reaction) ................................................................................ 37 
4.2.1.3.1.  Two step PCR ........................................................................................................ 38 
4.2.1.3.2.  Quantitative real time PCR (qPCR) ....................................................................... 38 
4.2.1.4.  Protein biochemical and immunological methods ....................................................... 39 
4.2.1.4.1.  Preparation of total cell lysates .............................................................................. 39 
4.2.1.4.2.  Determination of total protein concentration .......................................................... 39 
4.2.1.4.3.  Discontinous SDS-Polyacrylamidgelelectrophoresis (SDS-Page) ........................ 39 
4.2.1.4.4.  Western Transfer ................................................................................................... 40 
4.2.1.4.5.  Immunoblot ..............................