Mutational analysis of PhiC31 integrase to improve gene therapeutic applications [Elektronische Ressource] / von Raphael Liesner

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie
Lehrstuhl Virologie
Direktor: Prof. Dr. U. Koszinowski




Mutational analysis of PhiC31 integrase
to improve gene therapeutic applications


Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München














von
Raphael Liesner


1. Dezember 2009


































Eingereicht am: 1. Dezember 2009

Erster Gutacher: Prof. Dr. Heinrich Leonhardt
Zweiter Gutacher: PD Dr. Berit Jungnickel
Sonderberichterstatter: PD Dr. Anja Ehrhardt

Tag der mündlichen Prüfung: 2. November 2010
Table of contents

Table of contents..........................................................................................................................i

List of Tables..............................................................................................................................iv

List of Figures ............................................................................................................................. v

Abbreviations............................................................................................................................vii

Deutsche Zusammenfassung ..................................................................................................... 1

1. Introduction ............................................................................................................................ 2
1.1 Gene therapy ....................................................................................................................... 2
1.2 Gene transfer systems.......................................................................................................... 4
1.3 Non-viral vectors................................................................................................................. 6
1.3.1 Episomally persisting non-viral vectors........................................................................... 6
1.3.2 DNA recombination-mediated integration and excision by transposases and
recombinases ................................................................................................................. 7
1.4 PhiC31 integrase ............................................................................................................... 12
1.4.1 Recombination and integration efficiency ..................................................................... 12
1.4.2 Integration specificity and aberrant integration events .................................................. 14
1.4.3 Domain organisation and putative protein structure of the PhiC31 integrase................ 16
1.4.4 Applications for PhiC31 integrase ................................................................................. 20
1.4.5 Attempts to improve PhiC31 integrase integration efficacy .......................................... 20
1.5 Aims of this study ............................................................................................................. 22

2. Material ................................................................................................................................. 23
2.1 Laboratory equipment ....................................................................................................... 23
2.2 Chemicals .......................................................................................................................... 23
2.3 Enzymes ............................................................................................................................ 24
2.3.1 Restriction endonucleases .............................................................................................. 24
2.3.2 Other enzymes................................................................................................................ 24
2.4 Solutions, media and buffers............................................................................................. 25
2.4.1 Media, supplements and reagents for eukaryotic cell lines............................................ 27
2.5 Kits .................................................................................................................................... 28
2.6 Organisms.......................................................................................................................... 29
2.6.1 Bacteria........................................................................................................................... 29
2.6.2 Eukaryotic cells .............................................................................................................. 29
2.7 Oligonucleotides................................................................................................................ 30
2.8 Plasmids ............................................................................................................................ 33

i
3. Methods ................................................................................................................................. 35
3.1 General methods with eukaryotic cell culture................................................................... 35
3.1.1 Cultivation of eukaryotic cells ....................................................................................... 35
3.1.2 Storage of eukaryotic cells ............................................................................................. 35
3.1.3 Cell counting of cultured eukaryotic cells...................................................................... 35
3.1.4 Transfection of eukaryotic cells with plasmid DNA...................................................... 35
3.1.5 Transfection with siRNA and plasmid DNA and further applications .......................... 36
3.2 Process to select stable cell lines with integrated plasmid ................................................ 38
3.2.1 Quantification of integration events............................................................................... 39
3.2.2 Establishment of stably transfected cell lines ................................................................ 40
3.2.3 Isolation of genomic DNA from eukaryotic cells .......................................................... 40
3.2.4 Analysis of integration events by plasmid rescue .......................................................... 41
3.2.5 Estimation of the quantity of colonies dependent on the integrase plasmid
concentration ............................................................................................................... 42
3.3 Molecular biology techniques ........................................................................................... 43
3.3.1 Strain cultivation and storage of bacteria....................................................................... 43
3.3.2 Transformation of bacteria ............................................................................................. 43
3.3.3 Preparation of plasmid DNA.......................................................................................... 45
3.3.4 Polymerase chain reaction (PCR)................................................................................... 45
3.3.5 Design of a linker sequence and construction of a cloning vector pCS+NotI ............... 48
3.3.6 Restriction digestion of pDNA and gel electrophoresis................................................. 48
3.3.7 Isolation of DNA fragments from agarose gels ............................................................. 49
3.3.8 Dephosphorylation of DNA fragments .......................................................................... 49
3.3.9 Ligation of DNA fragments and vectors ........................................................................ 49
3.3.10 Determination of DNA concentration .......................................................................... 49
3.3.11 DNA sequencing and DNA alignments ....................................................................... 50
3.3.12 Analysis of RNA, isolation and reverse transcription into cDNA ............................... 50
3.3.13 Quantitative real-time PCR to determine relative DAXX knock down....................... 50
3.4 Fluorescence activated cell sorting (FACS)...................................................................... 51
3.5 Dual luciferase assay......................................................................................................... 52
3.6 Animal studies................................................................................................................... 54
3.6.1 Hydrodynamic tail vein injection................................................................................... 54
3.6.2 Measurement of alanine aminotransferase (ALT).......................................................... 54
3.6.3 Enzyme Linked Immunoabsorbent Assay (ELISA)....................................................... 54

4. Results .............................