SUMMARY ......................................................... 1
1 INTRODUCTION ................................................. 3
1.1 From recombinant DNA to genome engineering .............. 3
1.2 Site-specific recombination and key players ............. 4
Site-specific recombination and types of recombinases ... 4
Members of tyrosine recombinase family .................. 8
Cre/loxP ................................................ 9
Controlling the outcome of recombination ................ 9
loxP variants for broadening the Cre/loxP utility ...... 11
Other tyrosine recombinase systems ..................... 12
1.3 Applications of SSR .................................... 14
Temporal control of recombinase expression ............. 15
Tissue-specific knockouts .............................. 16
Cre-mediated gene activation and tissue-specific gene
repair ................................................. 17
Advanced applications of SSR in genome engineering ..... 17
Recombinase-mediated cassette exchange (RMCE) .......... 17
FLEx switch ............................................ 18
AIM OF THE THESIS .............................................. 21
2 RESULTS ..................................................... 22
2.1 Zre recombinase: Genetic approach ...................... 24
2.1.1 Choice of the source ............................ 24
2.1.2 Primer design and screen optimization ........... 24
2.1.3 Degenerative PCR screen and hits ................ 27
2.1.4 Retrieving full-length gene ..................... 28
2.1.5 Zre and Cre protein alignment ................... 30
2.1.6 Zre recombinase specificity ..................... 31
2.1.7 Zre target site ................................. 33
2.1.8 Activity of Zre and Cre on zox recombination
site ............................................ 35
2.1.9 Conclusions on Zre recombinase .................. 37
2.2 Vika recombinase: Rational prediction of SSR ........... 38
2.2.1 Identification of a new tyrosine recombinases ... 38
2.2.2 Vika and VCre, two predicted tyrosine
recombinases .................................... 40
2.2.3 Vika is a putative novel recombinase. Sequence
alignment ....................................... 40
2.2.4 Vika does not recombine target sites of known
recombinase systems ............................. 42
2.2.5 Prediction of Vika the target site .............. 44
2.2.6 Vika specifically recombines vox sites .......... 46
2.2.7 Catalytically inactive VikaR343F does not
recombine vox ................................... 49
2.2.8 Comparative analysis of different recombinase
systems in E. coli .............................. 50
2.2.9 Vika/vox is efficient and specific in
mammalian cells ................................. 51
2.2.10 Vika does not cause cytopathic effects in
mammalian cells ................................. 53
2.3 Utility of the rational approach: Universality and
Applications ........................................... 63
2.3.1 Eight more SSR systems predicted ................ 63
2.3.2 Organism-specific recombinase applications ...... 68
3 DISCUSSION .................................................. 71
3.1 Zre recombinase ........................................ 71
3.2 Novel site-specific recombinases for manipulation of
the genomes ............................................ 72
3.3 Specificity and activity of Vika ....................... 73
3.4 Application of novel recombinases ...................... 74
3.5 Vika as an alternative tool for "troublesome"
organisms/tissues ...................................... 76
3.6 Recombinase toxicity in mammals. Prospects for
non-toxic Vika ......................................... 78
4 MATERIALS AND METHODS ....................................... 81
4.1 Enzymes, chemicals, reagents and kits .................. 81
4.2 Synthetic oligonucleotides ............................. 81
4.3 Bacterial techniques ................................... 81
4.3.1 Bacterial strain ................................ 81
4.3.2 Maintenance and media ........................... 81
4.3.3 Arabinose induction ............................. 82
4.3.4 Preparation and transformation of competent
cells ........................................... 82
4.4 Recombinant DNA techniques ............................. 82
4.4.1 Preparation of plasmid DNA ...................... 82
4.4.2 Restriction enzyme digestion .................... 83
4.4.3 DNA gel electrophoresis ......................... 83
4.4.4 Ligation ........................................ 83
4.4.5 Polymerase Chain reaction (PCR) ................. 83
4.4.6 Sequencing of DNA ............................... 83
4.5 Cell Culture ........................................... 83
4.5.1 Materials ....................................... 83
4.5.2 Cell culturing and transfection ................. 84
4.6 Plasmids ............................................... 84
4.6.1 Construction of recombinase reporters pEVO ...... 84
4.6.2 Construction of integration assay plasmids ...... 84
4.6.3 Construction of mammalian recombinase
expression vectors .............................. 85
4.6.4 Construction of lacZ reporter vectors ........... 85
4.6.5 Construction of GFP reporter vectors ............ 85
4.6.6 Recombined versions of reporter plasmids ........ 85
4.6.7 Construction of retroviral vectors .............. 86
4.7 Recombination Assays ................................... 86
4.7.1 Recombination assay based on pEVO vector ........ 86
4.7.2 lacZ excision assay ............................. 87
4.7.3 Integration assay ............................... 87
4.7.4 GFP assay ....................................... 88
4.8 Biochemistry ........................................... 88
4.8.1 Preparation of the cell extract ................. 88
4.8.2 SDS-PAGE ........................................ 89
4.8.3 Immunoblotting (Western blot) ................... 89
4.9 Bioinformatics ......................................... 90
4.9.1 Protein and DNA searches, SeLOX ................. 90
4.9.2 Protein structure analysis and alignments ....... 90
4.9.3 Cryptic lox-like sites genome analysis .......... 90
4.10 Imaging ................................................ 90
4.11 FACs ................................................... 91
4.12 Retrovirus production and infection .................... 91
4.13 Growth inhibition assay ................................ 91
4.14 DNA damage evaluation: gammaH2AX assay ................. 92
APPENDIX ....................................................... 93
REFERENCES ..................................................... 94
ACKNOWLEDGEMENTS .............................................. 102
DECLARATION ................................................... 103
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