Novel cre-like site-specific recombinases for genome engineering:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 104 S. Ill., graph. Darst. |
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| adam_text | TABLE OF CONTENTS
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
13 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
23 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
33 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
3.7
BIOINFORMATIC PREDICTION ORRECOMDFNASES: FROM VLRA TO A PALETTE
ORRECOMDUIASES SO
HTTP://D-NB.INFO/1043679227
4. MATERIALS AND METHODS 81
4.1. ENZYMES, CHEMICALS, REAGENTS AND KITS 81
4.2. SYNTHETIC OLIGONUCLEOTIDES 81
43. 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
4.8.1. 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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| dewey-ones | 660 - Chemical engineering |
| dewey-raw | 660.65 |
| dewey-search | 660.65 |
| dewey-sort | 3660.65 |
| dewey-tens | 660 - Chemical engineering |
| discipline | Chemie / Pharmazie Biologie |
| format | Thesis Book |
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| spelling | Karimova, Madina 1986- Verfasser (DE-588)1031159053 aut Novel cre-like site-specific recombinases for genome engineering von Madina Karimova 2012 104 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dresden, Techn. Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025754622&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Karimova, Madina 1986- Novel cre-like site-specific recombinases for genome engineering |
| subject_GND | (DE-588)4113937-9 |
| title | Novel cre-like site-specific recombinases for genome engineering |
| title_auth | Novel cre-like site-specific recombinases for genome engineering |
| title_exact_search | Novel cre-like site-specific recombinases for genome engineering |
| title_full | Novel cre-like site-specific recombinases for genome engineering von Madina Karimova |
| title_fullStr | Novel cre-like site-specific recombinases for genome engineering von Madina Karimova |
| title_full_unstemmed | Novel cre-like site-specific recombinases for genome engineering von Madina Karimova |
| title_short | Novel cre-like site-specific recombinases for genome engineering |
| title_sort | novel cre like site specific recombinases for genome engineering |
| topic_facet | Hochschulschrift |
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