The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons:
Gespeichert in:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2013
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| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | Nebentitel: Der Rezeptor Guanylylzyklase Npr2 reguliert die Bifurkation der kranialen sensorischen Axone. |
| Beschreibung: | IX, 117 Bl. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Ter-Avetisyan, Gohar |d 1982- |e Verfasser |0 (DE-588)1042350094 |4 aut | |
| 245 | 1 | 0 | |a The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons |c by Gohar Ter-Avetisyan |
| 246 | 1 | 3 | |a Der Rezeptor Guanylylzyklase Npr2 reguliert die Bifurkation der kranialen sensorischen Axone |
| 264 | 1 | |c 2013 | |
| 300 | |a IX, 117 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Nebentitel: Der Rezeptor Guanylylzyklase Npr2 reguliert die Bifurkation der kranialen sensorischen Axone. | ||
| 502 | |a Berlin, Freie Univ., Diss., 2013 | ||
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
LIST OF ABBREVIATIONS IV
ABSTRACT VIII
ZUSAMMENFASSUNG IX
1. INTRODUCTION 1
1.1 AXONAL PATHFINDING 1
1.2 AXONAL BRANCHING 2
1.3 AXONAL PROJECTIONS OF DORSAL ROOT GANGLION (DRG) NEURONS 3
1.4 THE CGMP-DEPENDENT SIGNALING PATHWAY IN AXONAL BRANCHING 4
1.5 CHARACTERIZATION OF CNP, NPR2 AND CGKI 7
1.6 CRE AND FLP RECOMBINATION IN GENE TARGETING 8
1.6.1 THE INDUCIBLE CRE RECOMBINATION SYSTEM 10
1.6.2 INDUCIBLE CRE-MEDIATED REPORTER GENE EXPRESSION 11
1.6.3 SINGLE NEURON LABELING USING INDUCIBLE CRE REPORTER LINES 12
2. PREFACE AND OBJECTIVE 14
3. MATERIALS 16
3.1 BUFFERS, SOLUTIONS AND MEDIA 16
3.2 RESTRICTION ENZYMES 18
3.3 POLYMERASES 19
3.4 EMBRYONIC STEM (ES) CELLS 19
3.5 KITS 19
3.6 ANTIBIOTICS 19
3.7 BACTERIAL STRAINS 20
3.8 ANTIBODIES FOR IMMUNOHISTOCHEMISTRY 20
3.9 CHEMICALS 20
3.10 SPECIAL EQUIPMENT 22
4. METHODS 23
4.1 MOLECULAR BIOLOGY 23
4.1.1 BACTERIAL CULTURE 23
4.1.2 TRANSFORMATION OF E. COLI 24
4.1.3 CHEMICAL TRANSFORMATION OF E. COLI 24
4.1.4 TRANSFORMATION OF E. COLI BY ELECTROPORATION 24
4.1.5 HOMOLOGOUS RECOMBINATION IN E. COLI 25
4.1.6 PURIFICATION OF MINI-A DNA 25
4.1.7 INDUCTION OF MINI-A RECOMBINATION FUNCTION 26
4.1.8 E. COLI STRAINS EL250 AND EL350: CONFIRMATION OF FUNCTIONAL FLP
AND CRE TARGET
SITES 26
4.1.9 ISOLATION OF PLASMID DNA 27
4.1.10 ISOLATION OF BACTERIAL ARTIFICIAL CHROMOSOME (BAC) DNA 27
4.1.11 DETERMINATION OF DNA CONCENTRATION 27
4.1.12 RESTRICTION ENZYME DIGESTION OF PLASMID DNA 28
4.1.13 AGAROSE GEL ELECTROPHORESIS 28
I
HTTP://D-NB.INFO/1043487387
TABLE OF CONTENTS
4.1.14 ISOLATION OF DNA FROM AGAROSE GELS (GEL EXTRACTION) 28
4.1.15 POLYMERASE-CHAIN-REACTION (PCR) 29
4.1.16 PCR FOR MOLECULAR CLONING 29
4.1.17 BLUNTING OF DNA FRAGMENTS 30
4.1.18 DEPHOSPHORYLATION AND PHOSPHORYLATION 31
4.1.19 LIGATION 31
4.1.20 CONTROL OF THE DIRECTIONAL CLONING 32
4.2 MOUSE EMBRYONIC STEM CELL CULTURE AND ESTABLISHING THE MOUSE MODELS
NPR2LACZ
AND NPR2CREERT2 32
4.2.1 CULTURING OF FEEDER CELLS 33
4.2.2 CULTURING OF ES CELLS 34
4.2.3 ELECTROPORATION OF ES CELLS 34
4.2.4 SELECTION, PICKING AND CULTURING OF GENETICIN RESISTANT CLONES 35
4.2.5 EXTRACTION OF GENOMIC ES CELL DNA 36
4.2.6 SOUTHERN BLOT ANALYSIS OF HOMOLOGOUS RECOMBINATION 36
4.2.7 CULTURING AND EXPANDING ES CELL CLONES WITH THE CORRECT HOMOLOGOUS
RECOMBINATION 40
4.2.8 CONFIRMATION OF THE CORRECT HOMOLOGOUS RECOMBINATION 40
4.2.9 BLASTOCYST INJECTION AND GENERATION OF THE CHIMERA 40
4.2.10 GERMLINE TRANSMISSION, REMOVAL OF THE NEO-CASSETTE AND
ESTABLISHING THE MOUSE
STRAINS NPR2LACZ AND NPR2CREERT2 41
4.3 MOUSE GENOTYPING 41
4.3.1 GENOTYPING OF THE FLP-DELETER MOUSE STRAIN 42
4.3.2 GENOTYPING OF THE Z/AP MOUSE STRAIN 42
4.3.3 GENOTYPING OF THE NPR2LACZ MOUSE STRAIN 42
4.3.4 GENOTYPING OF THE NPR2(CN) MOUSE STRAIN 43
4.3.5 GENOTYPING OF THE NPR2CREERT2 MOUSE STRAIN 43
4.3.6 GENOTYPING OF THE MGFP MOUSE STRAIN 44
4.4 HISTOLOGY 45
4.4.1 DETECTION OF THE 3-GALACTOSIDASE ACTIVITY: X-GAL STAINING ON THE
WHOLE MOUNTS....45
4.4.2 CLEARING AND IMAGING OF THE WHOLE MOUNTS 46
4.4.3 DETECTION OF THE (3-GALACTOSIDASE ACTIVITY: X-GAL STAINING ON
CRYOSECTIONS 46
4.4.4 IMMUNOHISTOCHEMISTRY ON CRYOSECTIONS 47
4.4.5 DETECTION OF ALKALINE PHOSPHATASE (AP) ACTIVITY ON WHOLE MOUNTS
AND VIBRATOME
SECTIONS 48
4.5 ADMINISTRATION OF 4-HYDROXYTAMOXIFEN (OHT) AND TAMOXIFEN 49
4.6 MOUSE PERFUSION 49
4.7 STIMULATION OF DRG BY THE CGMP ANALOGUE 8-PCPT-CGMP AND CNP-22 50
4.8 WESTERN BLOT ANALYSIS OF DRG 50
4.9 DIL-LABELING OF THE DRG SENSORY AXONS 51
4.10 MAINTENANCE AND BREEDING OF MICE 51
5. RESULTS 52
5.1 GENERATION OF THE NPR2LACZ MOUSE LINE FOR MONITORING NPR2 EXPRESSION
52
5.1.1 CLONING STRATEGY 52
5.1.2 HOMOLOGOUS RECOMBINATION IN EMBRYONIC STEM (ES) CELLS 56
TABLE OF CONTENTS
5.1.3 BLASTOCYST INJECTION, CHIMERA PRODUCTION AND GERMLINE TRANSMISSION
57
5.2 VERIFICATION OF THE NPR2LACZ MOUSE LINE 58
5.2.1 MOLECULAR GENETIC ANALYSIS OF THE NPR2LACZ MOUSE 58
5.2.2 BIOCHEMICAL ANALYSIS OF THE NPR2LACZ MOUSE 59
5.2.3 PHENOTYPIC VERIFICATION OF THE NPR2LACZ MOUSE 61
5.3 EMBRYONIC EXPRESSION PATTERN OF NPR2 61
5.4 CO-LOCALIZATION OF NPR2 AND CGKIA IN NEURONAL CELLS OF EARLY MOUSE
EMBRYOS 66
5.5 EXPRESSION OF NPR2 IN POSTNATAL AND ADULT MOUSE BRAIN 70
5.6 GENERATION OF THE NPR2CREERT2 MOUSE LINE FOR SPARSE LABELING OF NPR2
EXPRESSING
NEURONS 72
5.6.1 CLONING STRATEGY 72
5.6.2 HOMOLOGOUS RECOMBINATION IN ES CELLS 75
5.6.3 BLASTOCYST INJECTION, CHIMERA PRODUCTION AND GERMLINE TRANSMISSION
76
5.6.4 ESTABLISHMENT OF THE NPR2CREERT2 MOUSE LINE 77
5.7 PHENOTYPIC VERIFICATION OF THE NPR2CREERT2 MOUSE 77
5.8 SPARSE NEURON LABELING USING THE NPR2CREERT2 MOUSE 78
5.8.1 PROOF-OF-PRINCIPLE: USING THE NPR2CREERT2 MOUSE FOR SINGLE AXON
LABELING AND
BRANCHING ANALYSIS 80
5.9 ANALYZING THE BRANCHING PATTERN OF CRANIAL SENSORY AXONS 82
5.9.1 VISUALIZATION OF AXON PROJECTIONS OF SINGLE CRANIAL SENSORY
NEURONS BY CROSSING OF
NPR2CREERT2 MOUSE WITH THE Z/AP REPORTER LINE 82
5.9.2 VISUALIZATION OF AXON PROJECTIONS OF SINGLE CRANIAL SENSORY
NEURONS BY CROSSING OF
NPR2CREERT2 MOUSE WITH THE MGFP REPORTER LINE 84
5.9.3 QUANTIFICATION OF BIFURCATION ERRORS OF CRANIAL SENSORY AXONS 86
5.10 ANALYSIS OF PHOSPHORYLATION TARGETS OF CGKIA 87
6. DISCUSSION 89
6.1 IDENTIFICATION OF NEURONAL SUBPOPULATIONS WITH A CO-LOCALIZATION OF
NPR2 AND CGKIA 91
6.2 ANALYZING THE CENTRAL PROJECTIONS OF NPR2-POSITIVE CRANIAL SENSORY
NEURONS BY A
GENETIC APPROACH AT A SINGLE NEURON LEVEL 93
6.3 THE NPR2-POSITIVE MTN NEURONS AND NEURONAL CELLS IN THE RHOMBOMERE 1
96
6.4 EXPRESSION OF NPR2 IN CRANIAL SENSORY GANGLIA OF DUAL ORIGIN 97
6.5 THE PHYSIOLOGICAL ROLE OF CNP-NPR2 SIGNALING AND INVOLVEMENT OF NPR2
IN A HUMAN
DISEASE 98
6.6 QUEST FOR PHOSPHORYLATION TARGET(S) OF THE CNP-NPR2-CGKIA PATHWAY 99
7. FUTURE PERSPECTIVES 101
8. REFERENCE LIST 102
9. CURRICULUM VITAE 111
10. APPENDIX 114
10.1 CLONING PLASMIDS 114
10.2 ANTIBODIES USED FOR WESTERN BLOTS OF DRG 117
|
| any_adam_object | 1 |
| author | Ter-Avetisyan, Gohar 1982- |
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| physical | IX, 117 Bl. Ill., graph. Darst. |
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| spelling | Ter-Avetisyan, Gohar 1982- Verfasser (DE-588)1042350094 aut The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons by Gohar Ter-Avetisyan Der Rezeptor Guanylylzyklase Npr2 reguliert die Bifurkation der kranialen sensorischen Axone 2013 IX, 117 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Nebentitel: Der Rezeptor Guanylylzyklase Npr2 reguliert die Bifurkation der kranialen sensorischen Axone. Berlin, Freie Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:kobv:188-fudissthesis000000095218-9 http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000095218 kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026742564&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ter-Avetisyan, Gohar 1982- The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons |
| subject_GND | (DE-588)4113937-9 |
| title | The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons |
| title_alt | Der Rezeptor Guanylylzyklase Npr2 reguliert die Bifurkation der kranialen sensorischen Axone |
| title_auth | The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons |
| title_exact_search | The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons |
| title_full | The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons by Gohar Ter-Avetisyan |
| title_fullStr | The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons by Gohar Ter-Avetisyan |
| title_full_unstemmed | The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons by Gohar Ter-Avetisyan |
| title_short | The receptor guanylyl cyclase Npr2 regulates the bifurcation of cranial sensory axons |
| title_sort | the receptor guanylyl cyclase npr2 regulates the bifurcation of cranial sensory axons |
| topic_facet | Hochschulschrift |
| url | http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000095218 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026742564&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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