Characterisation of the multivalent transcription factor CTCF:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English German |
| Published: |
2004
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Erlangen-Nürnberg, Univ., Diss., 2004. - Enth. Zsfssung in dt. und engl. Sprache |
| Physical Description: | III, 85 S. Ill. |
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| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2004 | ||
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Record in the Search Index
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| adam_text | Index
1 Summary 1
2 zusammenfassung 2
3 Introduction 3
3.1 Insulators as regulatory elements in the eukaryotic genome 3
3.2 Enhancer-blocking mediated by insulators 5
3.3 The multivalent vertebrate transcription factor CTCF 8
3.3.1 Structural overview of CTCF 8
3.3.2 The diverse activities of the transcriptional regulator CTCF 10
3.3.2.1 Transcriptional activation 10
3.3.2.2 Transcriptional repression 10
3.3.2.3 The role of CTCF for the chicken 5 HS4 insulator activity 11
3.4 The Tet-system 12
3.5 Scope of the thesis 13
4 Results 14
4.1 Enhancer-blocking activity of CTCF-based transregulators 14
4.1.1 Construction of an SV40-based enhancer blocking assay 14
4.1.1.1 Linker-design between the TetR-CTCF moieties and its influence
on enhancer-blocking activity 14
4.1.1.2 Number of tet operators necessary for sc TetR-CTCF mediated
enhancer-blocking 17
4.1.1.3 Influence of the position of a (tetOih element on sc TetR-CTCF
mediated enhancer-blocking activity 18
4.1.1.4 Influence of the distance of (letOi)i to the enhancer on
sc TetR-CTCF mediated enhancer-blocking activity 20
4.1.2 Characterisation of CTCF-domains in an SV40-based
enhancer-blocking assay 22
4.1.2.1 Enhancer-blocking activity of different CTCF domains 22
4.1.2.2 Positional enhancer-blocking activity of active CTCF domains 25
4.1.2.3 Influence of active CTCF domains on an SV40 minimal promoter. 26
4.1.3 Mapping of repression activity within the N-terminal domain of CTCF 27
4.1.3.1 Rough mapping of repression activity within the N-terminus
of CTCF 27
4.1.3.2 Fine mapping of repression activity within the repression motif
nCTCF(130-190) 30
- i-
Index
4.1.3.3 Influence of the fine mapped N-terminal repression motif
CTCF(150-170) on an SV40 minimal promoter 33
4.2 Transcriptional repression mediated by short co-repressor recruiting motifs 34
4.3 HeLa Flp-In™ Host Cell Lines 37
4.3.1 Generation and Characterisation of HeLa Flp-In™ Host Cell Lines 38
4.3.1.1 Generation of stable HeLa Flp-In™ Host Cell Lines 38
4.3.1.2 Characterisation of primary HeLa Flp-In™ Host Cell Clones 38
4.3.2 Generation and Characterisation of HeLa Flp-In™ Reporter Cell Lines 41
4.3.2.1 Generation and characterisation of the HF263 /264
Reporter Cell Lines 41
4.3.2.2 Generation and characterisation of the HF275 Reporter Cell Line 44
5 Discussion 46
5.1 Construction of an SV40-based enhancer blocking assay 46
5.1.1 Enhancer-blocking activity of CTCF-based transregulators 46
5.1.1.1 The fusion structure between TetR and CTCF has only little influence
on enhancer-blocking activity 47
5.1.1.2 A minimum number of three tet operators is necessary for
sc TetR-CTCF mediated enhancer-blocking 47
5.1.1.3 sc TetR-CTCF mediated enhancer-blocking activity is position
independent 48
5.1.1.4 CTCF subdomains repress luciferase activity in a position
independent manner 49
5.1.2 Mapping of a repression motif within the N-terminal domain of CTCF 50
5.2 Transcriptional repression mediated by short co-repressor recruiting domains 55
5.3 Generation of a stably integrated enhancer-blocking test system 56
5.3.1 Generation and Characterisation of HeLa Flp-In™ Host Cell Lines 56
5.3.2 Generation and Characterisation of HeLa Flp-In™ Reporter Cell Lines 57
6 Materials and Methods 60
6.1 Materials 60
6.1.1 Chemicals 60
6.1.2 Auxiliary Materials 61
6.1.3 Instruments 61
6.1.4 Enzymes, Proteins and Size markers 62
6.1.5 Commercially available systems ( kits ) 62
6.1.6 Oligonucleotides 62
-ii- i
i
Index
6.1.7 Plasmids 64
6.1.8 Antibodies 70
6.1.9 Bacteria 70
6.1.10 Cell lines 70
6.2 Media, Buffers and Solutions 71
6.2.1 Bacterial growth media 71
6.2.2 Cell culture media 71
6.2.3 Buffers and solutions 72
6.3 General Methods 73
6.3.1 General methods 73
6.3.2 Cell culture 73
6.3.2.1 Cultivation of HeLa cell lines 73
6.3.2.2 Cell counts 73
6.3.2.3 Long term storage of cell lines 74
6.3.3 Methods for transfection and characterisation of cell lines 74
6.3.3.1 Transient transfection of HeLa cells using Lipofectamine™ 74
6.3.3.2 Transient transfection of HeLa cells using PerFectin™ 74
6.3.3.3 Harvesting of HeLa cells 74
6.3.3.4 Determination of protein concentrations 74
6.3.3.5 P-galactosidase assay 75
6.3.3.6 Luciferase assay 75
6.3.3.7 Stable transfection of HeLa cells 75
6.3.3.8 In situ p-galactosidase assay 76
6.3.3.9 Fluorescence activated cell sorting (FACS) 76
6.3.4 Western blot analysis 76
6.3.5 Southern blot analysis 77
6.3.5.1 Random primed labelling of DNA-probe 77
6.3.5.2 Preparation, separation and transfer of genomic DNA 77
6.3.5.3 Detection / Visualisation of probe-target hybrids 78
6.3.6 Methods for nucleic acid purification 78
7 Literature 85
|
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| illustrated | Illustrated |
| indexdate | 2024-07-09T19:23:04Z |
| institution | BVB |
| language | English German |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-010738103 |
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| physical | III, 85 S. Ill. |
| publishDate | 2004 |
| publishDateSearch | 2004 |
| publishDateSort | 2004 |
| record_format | marc |
| spelling | Drüppel, Lars Verfasser aut Characterisation of the multivalent transcription factor CTCF von Lars Drüppel Characterization of the multivalent transcription factor CTCF 2004 III, 85 S. Ill. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2004. - Enth. Zsfssung in dt. und engl. Sprache Erlangen-Nürnberg, Univ., Diss., 2004 Protein CTCF Protein CTCF (DE-588)4605808-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Protein CTCF (DE-588)4605808-4 s DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010738103&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Drüppel, Lars Characterisation of the multivalent transcription factor CTCF Protein CTCF Protein CTCF (DE-588)4605808-4 gnd |
| subject_GND | (DE-588)4605808-4 (DE-588)4113937-9 |
| title | Characterisation of the multivalent transcription factor CTCF |
| title_alt | Characterization of the multivalent transcription factor CTCF |
| title_auth | Characterisation of the multivalent transcription factor CTCF |
| title_exact_search | Characterisation of the multivalent transcription factor CTCF |
| title_full | Characterisation of the multivalent transcription factor CTCF von Lars Drüppel |
| title_fullStr | Characterisation of the multivalent transcription factor CTCF von Lars Drüppel |
| title_full_unstemmed | Characterisation of the multivalent transcription factor CTCF von Lars Drüppel |
| title_short | Characterisation of the multivalent transcription factor CTCF |
| title_sort | characterisation of the multivalent transcription factor ctcf |
| topic | Protein CTCF Protein CTCF (DE-588)4605808-4 gnd |
| topic_facet | Protein CTCF Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010738103&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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