Self-inactivating gammaretroviral vectors for the gene therapy of chronic granulomatous disease [Elektronische Ressource] / von Bibiana Moreno Carranza

Bibi Naceri

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 52
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Self-inactivating gammaretroviral vectors for the
gene therapy of Chronic Granulomatous Disease



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich Biowissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main



von
Bibiana Moreno Carranza
aus Barcelona, Spain

Frankfurt 2008











Vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe-Universität als
Dissertation angenomen.





Dekan: Prof. Dr. Volker Müller

Gutachter: Prof. Dr. Anna Starzinski-Powitz
Prof. Dr. Bernd Groner

Datum der Disputation: ………………………….












a mis padres, hermana y abuela



“Caminante, son tus huellas
el camino y nada más;
caminante, no hay camino,
se hace camino al andar.

Al andar se hace camino
y al volver la vista atrás
se ve la senda que nunca
se ha de volver a pisar.

Caminante no hay camino
sino estelas en la mar...”
Antonio Machado

TABLE OF CONTENTS I
TABLE OF CONTENTS
A. INTRODUCTION ................................................................................................ 1
A.1 Chronic Granulomatous Disease ............................................................................................................... 1
A.1.1 Clinical manifestations ........................................................................................................................ 1
A.1.2 The NADPH oxidase enzyme complex................................................................................................ 1
A.1.3 Regulation of the oxidase assembly..................................................................................................... 6
A.1.4 Molecular defects................................................................................................................................. 8
A.1.5 Current therapy for CGD ..................................................................................................................... 9
A.2 Gene therapy............................................................................................................................................. 10
A.2.1 Gene transfer vector systems and their application............................................................................ 11
A.2.2 Retroviral vectors............................................................................................................................... 13
A.2.3 Success and risks of gene therapy...................................................................................................... 21
A.3 Gene therapy for CGD ............................................................................................................................. 23
A.4 Aims of the present study ......................................................................................................................... 27
B. MATERIALS AND METHODS....................................................................... 28
B.1 Equipment and Necessary Materials....................................................................................................... 28
B.2 Materials for Molecular Biological Experiments ................................................................................... 28
B.2.1 Chemicals, Enzymes and Reagents for Molecular Biological Experiments....................................... 28
B.2.2 Antibodies (Ab) for Molecular Biological Methods (Western Blot) ................................................. 30
B.2.3 Oligonucleotides................................................................................................................................ 30
B.2.4 Bacterial E.Coli strains ...................................................................................................................... 32
B.3 Molecular biological methods .................................................................................................................. 32
B.3.1 Phenol/Chloroform/Isoamyl alcohol extraction of nucleic acids ....................................................... 32
B.3.2 Ethanol (or 2-propanol) precipitation of nucleic acids ...................................................................... 33
B.3.3 Determination of nucleic acid concentration ..................................................................................... 33
B.3.4 Restriction endonuclease digestion of DNA...................................................................................... 33
B.3.5 Removal of 5’ phosphate groups of DNA (Antarctic Phosphatase)................................................... 33
B.3.6 Blunting ends reaction (DNA Polymerase I, Large (Klenow) Fragment).......................................... 33
B.3.7 Size separation of nucleic acids by agarose gel electrophoresis ........................................................ 34
B.3.8 Isolation/ purification of DNA fragments from agarose gels ............................................................. 34
B.3.9 DNA fragment ligation ...................................................................................................................... 34
B.3.10 Topo cloning kit ................................................................................................................................ 34
B.3.11 Chemical transformation of E.Coli .................................................................................................... 35
B.3.12 Plasmid DNA preparation.................................................................................................................. 35
B.3.13 Nucleic Acid isolation from cultured cells......................................................................................... 36
B.3.14 Polymerase Chain Reaction (PCR).................................................................................................... 36
B.3.15 Reverse transcription polymerase chain reaction (RT-PCR) ............................................................. 37
B.3.16 Site-Directed Mutagenesis................................................................................................................. 38
B.3.17 Preparation of high specific activity DNA probe............................................................................... 38
B.3.18 Southern Blot..................................................................................................................................... 39
B.3.19 Northern Blot..................................................................................................................................... 40
B.3.20 Western Blot...................................................................................................................................... 41
B.4 Specific equipment for cell culture .......................................................................................................... 43
B.5 Materials for cell culture.......................................................................................................................... 43
B.5.1 Chemicals and Reagents for Cell Culture .......................................................................................... 43
B.5.2 Antibodies for FACS analysis ........................................................................................................... 44
B.5.3 Cell lines............................................................................................................................................ 44 TABLE OF CONTENTS II
B.6 Cell culture methods................................................................................................................................. 45
B.6.1 Common cell culture.......................................................................................................................... 45
B.6.2 Thawing and freezing cells ................................................................................................................ 45
B.6.3 Assessing cell viability by Trypan Blue exclusion............................................................................. 46
B.6.4 Retroviral particle production via calcium phosphate Ca (PO4) mediated transfection................... 46 3 2
B.6.5 Titer determination and transduction of target cells........................................................................... 47
B.6.6 Flow cytometry.................................................................................................................................. 47
B.6.7 Cell differentiation............................................................................................................................. 47
B.6.8 Magnetic Cell Sorting........................................................................................................................ 48
B.6.9 Cytochrome C assay .......................................................................................................................... 48
B.6.10 DHR assay ...................................................................................................