The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | German |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-274107 Inhaltsverzeichnis |
| Beschreibung: | 117 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells |c von Simone Krinner |
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Datensatz im Suchindex
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|---|---|
| adam_text | THE IMPACT OF INTRAGENIC CPG CONTENT ON
EPIGENETIC CONTROL OF TRANSGENE EXPRESSION
IN MAMMALIAN
CELLS
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES
DER NATURWISSENSCHAFTEN (DR. RER. NAT.)
DER FAKULTAT FUR BIOLOGIE UND VORKLINISCHE MEDIZIN
DER UNIVERSITAT REGENSBURG
VORGELEGT VON
SIMONE KRINNER
AUS STRAUBING
IM JAHR 2012
HTTP://D-NB.INFO/1048211312
1 ZUSAMMENFASSUNG 1
2 ABSTRACT 3
3 INTRODUCTION 5
3.1 EUKARYOTIC GENE TRANSCRIPTION 5
3.2 CHROMATIN 7
3.2.1 THE NUCLEOSOME 7
3.2.2 CHROMATIN ORGANIZATION 8
3.3 TRANSCRIPTIONAL CONTROL BY CHROMATIN 10
3.3.1 HISTONE MODIFICATIONS 10
3.3.2 CHROMATIN REMODELING 13
3.3.3 SEQUENCE DEPENDENT NUCLEOSOME POSITIONING 14
3.4 CYTOSINE GUANINE DINUCLEOTIDES 15
3.4.1 CPG METHYLATION 15
3.4.2 UNMETHYLATED CPG DINUCLEOTIDES 16
3.4.3 GENE CONTROL MECHANISMS DIRECTED BY CPG DINUCLEOTIDES 17
3.5 TRANSGENE EXPRESSION .- 19
3.5.1 VIRAL VECTOR-BASED TRANSGENE EXPRESSION 20
3.5.2 PLASMID-BASED TRANSGENE EXPRESSION 21
3.6 OVERVIEW OF PRECEDING CPG STUDIES 23
3.6.1 THE MODEL GENES HGFP AND MMIP-IA 23
3.6.2 IMPACT OF INTRAGENIC CPG CONTENT OF HGFP AND MMIP-IA ON GENE
EXPRESSION 25
3.7 AIM OF THE STUDY 27
4 RESULTS 28
4.1 CPG-DEPENDENT DIFFERENTIAL TRANSGENE EXPRESSION USING MAMMALIAN
FLP-IN CELLS 28
4.1.1 LONG-TERM HGFP EXPRESSION IN THE PRESENCE OR ABSENCE OF SELECTION
PRESSURE 30
4.1.2 SORTING OF CHO FLP-IN CELLS ACCORDING TO HGFP EXPRESSION LEVELS 33
4.1.3 RELATIVE COPY NUMBER AND METHYLATION STATUS OF HGFP IN CORRELATION
TO EXPRESSION LEVELS 34
4.1.4 IMPACT OF INTRAGENIC CPG DINUCLEOTIDES ON CHROMATIN STRUCTURE 40
4.1.5 INFLUENCE OF INTRAGENIC CPG DINUCLEOTIDES ON RNAPII OCCUPATION 47
4.1.6 IMPACT OF INTRAGENIC CPG DISTRIBUTION ON GENE EXPRESSION IN HGFP
50
4.2 CPG-DEPENDENT DIFFERENTIAL TRANSGENE EXPRESSION IN MURINE EMBRYONIC
CARCINOMA CELLS P19 52
.4.2.1 GENERATION OF SIN-LVS INCORPORATING HGFP VARIANTS 53
4.2.2 LONG-TERM EXPRESSION OF HGFP VARIANTS IN P19 CELLS USING DIFFERENT
PROMOTERS 54
4.2.3 PARTIAL PREVENTION OF HGFP SILENCING IN P19 CELLS BY DNMT
INHIBITION 58
5 DISCUSSION 60
5.1 EVOLUTION OF CPG FREQUENCY IN THE MAMMALIAN GENOME 60
5.2 CPG DINUCLEOTIDE USAGE IS PIVOTAL FOR TRANSGENE EXPRESSION 61
5.3 INTRAGENIC CPG ABUNDANCE DETERMINES EXPRESSION LEVELS OF HGFP AND
MMIP-IA 61
5.4 INTRAGENIC CPG DINUCLEOTIDES CONFER NO DISADVANTAGE FOR LONG-TERM
EXPRESSION
IN MAMMALIAN FLP-IN CELLS 63
5.5 INTRAGENIC CPG DINUCLEOTIDES CAUSE INCREASED DNA METHYLATION RATES,
WHEREAS LOW CPG CONTENT
PROMOTES TRANSGENE LOSS 64
5.6 INTRAGENIC CPG DINUCLEOTIDES ALTER CHROMATIN STRUCTURE 65
5.6.1 CHROMATIN DENSITY OF HGFP TRANSGENES IS AFFECTED BY INTRAGENIC CPG
DINUCLEOTIDES
AND GROWTH CONDITIONS IN VIVO 65
5.6.2 CHROMATIN DENSITY OF MMIP-TA TRANSGENES IS INCREASED UPON CPG
DEPLETION IN VIVO 66
5.6.3 CPG DINUCLEOTIDES IN HGFP AFFECT NUCLEOSOME POSITIONING ABILITIES
IN VITRO 67
5.7 INTRAGENIC CPG DINUCLEOTIDES INCREASE TRANSCRIPTION ELONGATION OF
MMIP-IU 68
5.8 GENE EXPRESSION BENEFITS FROM TSS-PROXIMITY OF INTRAGENIC CPG
DINUCLEOTIDES 68
5.9 CPG FREQUENCY AND TYPE OF PROMOTER DETERMINES TRANSGENE STABILITY IN
PLURIPOTENT STEM CELLS P19 69
5.9.1 CMV- AND EF-IOC-PROMOTER-MEDIATED HGFP EXPRESSION IS GRADUALLY
SILENCED IN P19 CELLS 70
5.9.2 A2UCOE CONFERS STABLE HGFP EXPRESSION IN P19 CELLS AND PREVENTS
HGFP REPRESSION
UPON INTRAGENIC CPG DEPLETION 71
5.9.3 DNMT INHIBITION PARTLY PREVENTS HGFP SILENCING IN P19 CELLS
DEPENDING ON PROMOTER USAGE 72
5.10 PROPOSED CPG-MEDIATED TRANSCRIPTIONAL CONTROL MECHANISM AND OUTLOOK
73
6 MATERIALS 75
6.1 CELL LINES 75
6.2 BACTERIAL STRAINS 75
6.3 MEDIA AND SUPPLEMENTS 75
6.4 KITS 76
6.5 BUFFERS AND REAGENTS 76
6.6 PLASMIDS 79
6.7 OLIGONUCLEOTIDES 79
6.8 CHEMICALS, ENZYMES AND MATERIALS 80
7 METHODS 81
7.1 CULTIVATION OF EUKARYOTIC CELLS 81
7.1.1 MAINTENANCE OF CELL LINES 81
7.1.2 TRANSIENT TRANSFECTIONS 81
7.1.3 ESTABLISHMENT OF PLASMID-BASED STABLE CELL LINES 81
7.1.4 LENTIVIRAL VECTOR (LV) PREPARATION AND TRANSDUCTION OF CELL LINES
82
7.2 CULTIVATION OF PROKARYOTIC CELLS 82
7.3 DNA METHODS 82
7.3.1 ISOLATION OF GENOMIC DNA 82
7.3.2 DNA QUANTIFICATION 83
7.3.3 AGAROSE GEL ELECTROPHORESIS 83
7.3.4 DNA PURIFICATION FROM AGAROSE GELS 83
7.3.5 IN VITRO METHYLATION 83
7.3.6 BISULFITE CONVERSION AND SEQUENCE ANALYSIS 83
7.4 POLYMERASE CHAIN REACTION (PCR) 84
7.4.1 QUANTITATIVE PCR/REAL-TIME PCR 84
7.4.2 DNA SEQUENCING 85
7.5 PLASMID CONSTRUCTION 85
7.5.1 LIGATION 85
7.5.2 TRANSFORMATION OF E.COLI 85
7.5.3 PREPARATION OF PLASMID DNA 85
7.5.4 CLONING OF HGFP CHIMERA 86
7.5.5 CLONING OF LENTIVIRAL TRANSGENE VECTORS 86
7.6 PROTEIN METHODS 86
7.6.1 DETERMINATION OF PROTEIN AMOUNT ACCORDING TO BRADFORD 86
7.6.2 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 86
7.6.3 FLOW CYTOMETRY 87
7.7 FORMALDEHYDE-ASSISTED ISOLATION OF REGULATORY ELEMENTS (FAIRE) 87
7.8 CHROMATIN IMMUNOPRECIPITATION (CHIP) 88
7.9 ANALYSIS OF RECONSTITUTED MONONUCLEOSOMES IN VITRO 89
7.9.1 AMPLIFICATION OF CPG FRAGMENTS FOR NUCLEOSOME RECONSTITUTIONS 89
7.9.2 NUCLEOSOME ASSEMBLY BY SALT DIALYSIS 89
7.9.3 ANALYSIS OF MONONUCLEOSOMES BY NATIVE PAGE 90
8 REFERENCE LIST 91
9 APPENDIX NO
9.1 LIST OF ABBREVIATIONS 110
9.2 SEQUENCES U5
9.2.1 MURINE MLP-IA VARIANTS 115
9.2.2 HUMANIZED GFP VARIANTS 116
10 DANKSAGUNG 117
|
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| spelling | Krinner, Simone Verfasser aut The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells von Simone Krinner 2012 117 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss. Gentransfer (DE-588)4194102-0 gnd rswk-swf Dinucleotide (DE-588)4150007-6 gnd rswk-swf CpG-Inseln (DE-588)4584729-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Gentransfer (DE-588)4194102-0 s CpG-Inseln (DE-588)4584729-0 s Dinucleotide (DE-588)4150007-6 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-274107 http://epub.uni-regensburg.de/27410/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-274107 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027035202&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Krinner, Simone The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells Gentransfer (DE-588)4194102-0 gnd Dinucleotide (DE-588)4150007-6 gnd CpG-Inseln (DE-588)4584729-0 gnd |
| subject_GND | (DE-588)4194102-0 (DE-588)4150007-6 (DE-588)4584729-0 (DE-588)4113937-9 |
| title | The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells |
| title_auth | The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells |
| title_exact_search | The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells |
| title_full | The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells von Simone Krinner |
| title_fullStr | The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells von Simone Krinner |
| title_full_unstemmed | The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells von Simone Krinner |
| title_short | The impact of intragenic CpG content on epigenetic control of transgene expression in mammalian cells |
| title_sort | the impact of intragenic cpg content on epigenetic control of transgene expression in mammalian cells |
| topic | Gentransfer (DE-588)4194102-0 gnd Dinucleotide (DE-588)4150007-6 gnd CpG-Inseln (DE-588)4584729-0 gnd |
| topic_facet | Gentransfer Dinucleotide CpG-Inseln Hochschulschrift |
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