Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | IX, 102 Bl. Ill., graph. Darst. |
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| 100 | 1 | |a Kriegmair, Maximilian |d 1987- |e Verfasser |0 (DE-588)1049742745 |4 aut | |
| 245 | 1 | 0 | |a Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis |c von Maximilian Kriegmair |
| 264 | 1 | |c 2014 | |
| 300 | |a IX, 102 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 502 | |a München, Univ., Diss., 2014 | ||
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
TABLE OF CONTENTS V
ZUSAMMENFASSUNG VIII
SUMMARY. IX
1 INTRODUCTION 1
1.1 CLINICAL BACKGROUND AND OBJECTIVES ........... 1
1.2 THE AFRICAN CLAWED FROG
XENOPUS LAEVIS*
....... 2
1.2.1 XENOPUS LEAVIS AS MODEL ORGANISM 2
1.2.2 THE LIFE CYCLE OF XENOPUS LEAVIS 3
1.2.3 METHODICAL CONCEPTS IN XENOPUS LEAVIS RESEARCH 4
1.3 DEVELOPMENT OF THE HEART 6
1.3.1 MORPHOLOGICAL DEVELOPMENT OF THE HEART IN XENOPUS LAEVIS 6
1.3.2 MOLECULAR MECHANISMS OF HEART DEVELOPMENT 8
1.3.2.1 MESODERM INDUCTION 8
1.3.2.2 INDUCTION OF CARDIAC TISSUE. 9
1.3.2.3 DIFFERENTIATION OF CARDIAC PROGENITORS 10
1.3.2.4 THE CONCEPT OF PRIMARY AND SECONDARY HEART FIELDS - 12
1.4 THE CARDIOGENIC TRANSCRIPTION FACTOR
MESPL
... 13
1.4.1 BHLH TRANSCRIPTION FACTORS 13
1.4.2 MESPL - A GATEKEEPER OF CARDIOGENESIS 14
1.4.3 MESP RELATED GENES IN XENOPUS LAEVIS 17
2 METHODS
AND MATERIAL 19
2.1 LABORATORY EQUIPMENT *.*19
2.2 REAGENTS .......... 19
2.2.1 CHEMICALS 19
2.2.2 KITS 20
2.2.3 ENZYMES 20
2.2.4 BACTERIA 20
2.3 DNA TEMPLATES 20
2.3.1 PLASMID VECTORS FOR CLONING 20
2.3.2 PLASMIDS FOR IN VITRO TRANSCRIPTION 21
2.3.3 PLASMIDS FOR IN SITU HYBRIDIZATION PROBES 21
2.4 PRIMERS............ .... ..... ....... ..........* 21
2.4.1 PRIMERS FOR MESPB AND A5 UTR-MESPO CLONING 21
2.4.2 PRIMERS FOR QRT-PRC 22
V
HTTP://D-NB.INFO/1049742818
TABLE OF CONTENTS
2.5 MORPHOLINO OLIGONUCLEOTIDES * 22
2.6 ANTIBODIES ... 23
2.7 MOLECULAR BIOLOGICAL METHODS... .... ...... 24
2.7.1 SOLUTIONS 24
2.7.2 DNA TECHNIQUES 25
2.7.2.1 AGAROSE GEL ELECTROPHORESIS 25
2.7.2.2 ISOLATION OF DNA FRAGMENTS FROM AGAROSE GELS 26
2.7.2.3 CONCENTRATION MEASUREMENT AND PURITY CONTROL 26
2.7.2.4 RESTRICTION DIGESTION * 26
2.7.2.5 ISOLATION OF PLASMID DNA FROM E. COLI 26
2.7.2.6 DNA SEQUENCING * 26
2.7.2.7 CLONING OF PSP6-GLOBIN-MESPA-IRES-EGFP 26
2.7.2.8 CLONING OF PSP6-GLOBIN-MESPFC-IRES-EGFP 27
2.7.3 RNA TECHNIQUES 27
2.7.3.1 AGAROSE GEL ELECTROPHORESIS 27
2.7.3.2 CONCENTRATION MEASUREMENT AND PURITY CONTROL - 27
2.7.3.3 RNA EXTRACTION FROM ANIMAL TISSUE 27
2.7.3.4 RNA PURIFICATION 28
2.7.3.5 REVERSE TRANSCRIPTION FOR QRT-PCR 28
2.7.3.6 IN VITRO TRANSCRIPTION OF MESSENGER RNA 28
2.7.3.7 IN VITRO TRANSCRIPTION OF DIGOXYGENIN-LABELED RNA PROBES
............. 29
2.7.3.8 RNA IN SITU HYBRIDIZATION 30
2.7.4 POLYMERASE CHAIN REACTION 31
2.7.4.1 STANDARD POLYMERASE CHAIN REACTION - 31
2.7.4.2 QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION 31
2.8 PROTEIN TECHNIQUES..* - 32
2.8.1 SOLUTIONS 32
2.8.2 IN VITRO TRANSCRIPTION AND TRANSLATION 32
2.8.3 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS 32
2.9 HISTOLOGICAL TECHNIQUES 33
2.9.1 SOLUTIONS 33
2.9.2 LACZ STAINING 33
2.10 EMBRYOLOGICAL METHODS * * * 34
2.10.1 SOLUTIONS AND MATERIAL 34
2.10.2 LABORATORY ANIMALS 34
2.10.3 FERTILIZATION AND CULTIVATION OFXENOPUS LEAVIS EMBRYOS 34
2.10.4 MICROINJECTION 35
2.10.5 TISSUE EXPLANTS 36
VI
TABLE OF CONTENTS
3 RESULTS 38
3.1 EXPRESSION ANALYSIS OF
MESP
RELATED GENES- 38
3.1.1 SPATIAL EXPRESSION PATTERNS OF MESP RELATED GENES DURING
GASTRULATION 38
3.1.2 EXPRESSION OF MESP RELATED GENES DURING NEURULA AND TAILBUD STAGES
41
3.1.3 DEVELOPMENTAL MRNA PROFILE OF MESP RELATED GENES 42
3.2 OVEREXPRESSION OF
MESP
RELATED GENES ......................... 44
3.2.1 MESP RELATED GENES DRIVE ECTOPIC HEART MARKER EXPRESSION 44
3.2.1.1 LINEAGE TRACING OF HEART PRECURSORS * * 44
3.2.1.2 RNA OVEREXPRESSION 45
3.2.1.3 DNA OVEREXPRESSION 47
3.2.2 BIOLOGICAL ACTIVITY OF MESP RELATED GENES IN THE ANIMAL CAP ASSAY
50
3.2.2.1 INDUCTION OF CARDIAC MARKERS BY MESP RELATED GENES IN ANIMAL
CAPS SO
3.2.2.2 INDUCTION OF NKX2.S IN ANIMAL CAP CONJUGATES S3
3.3 MORPHOLINO BASED KNOCKDOWN STUDIES 54
3.3.1 MORPHOLINO DESIGN AND VALIDATION 54
3.3.2 HIERARCHIC RELATIONSHIP OF MESPA AND MESPO DURING GASTRULATION 56
3.3.2.1 MESPA IS REQUIRED FOR ITS OWN EXPRESSION 56
3.3.2.2 MESPA DEFICIENT EMBRYOS LACK MESPO EXPRESSION 57
3.3.3 IN THE ORGANIZER MESODERM DISSIMILAR MORPHOLINOS CAUSE A HEARTLESS
PHENOTYPE : 58
3.3.4 MESPA KNOCKDOWN PREVENTS DKKL MEDIATED HEART INDUCTION IN THE
VMZ.. 60
3.3.5 UNILATERAL MESPA KNOCKDOWN ABOLISHES HEART MARKER EXPRESSION 62
3.3.6 VALIDATION OF THE IN VIVO ACTIVITY OF MESPB AND MESPO MORPHOLINO
65
3.3.7 MESP RELATED GENES ARE NOT REQUIRED FOR EMBRYONIC PATTERING 67
3.3.8 FUNCTIONAL REDUNDANCY AMONG MESP RELATED GENES 68
3.3.9 MESPA MORPHANTS LACK TNN/3 EXPRESSION 70
3.3.10 MESPA DEFICIENT CELLS CANNOT CONTRIBUTE TO CARDIAC TISSUE 71
4 DISCUSSION 73
4.1 DEVELOPMENTAL EXPRESSION OF
MESP
RELATED GENES IN
X. LEAVIS
...................... 73
4.2 THE CARDIOGENIC ACTIVITY OF
MESP
RELATED GENES 7 6
4.3 MESPA ACTS UPSTREAM FROM MESPO AND ENHANCES ITS OWN EXPRESSION ....
79
4.4 IMPAIRED ORGANIZER MESODERM RESULTS IN HEARTLESS
PHENOTYPE................ 81
4.5 MESPA KNOCKDOWN IN
DKKL
INJECTED VENTRAL MARGINAL ZONE EXPLANTS 83
4.6
MESPA
IS REQUIRED FOR NORMAL HEART DEVELOPMENT 84
4.7
MESPB
AND
MESPO
CANNOT COMPENSATE FOR MESPA KNOCKDOWN ...._. 86
4.8 MESPA -
XENOPUS
HOMOLOGUE OF MAMMALIAN MESPL AND GATEKEEPER OF
CARDIOGENESLS 87
ABBREVIATIONS 89
REFERENCES 92
VII
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| language | English |
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| physical | IX, 102 Bl. Ill., graph. Darst. |
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| spelling | Kriegmair, Maximilian 1987- Verfasser (DE-588)1049742745 aut Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis von Maximilian Kriegmair 2014 IX, 102 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2014 (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=027235437&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kriegmair, Maximilian 1987- Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis |
| subject_GND | (DE-588)4113937-9 |
| title | Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis |
| title_auth | Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis |
| title_exact_search | Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis |
| title_full | Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis von Maximilian Kriegmair |
| title_fullStr | Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis von Maximilian Kriegmair |
| title_full_unstemmed | Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis von Maximilian Kriegmair |
| title_short | Functional analysis of Xenopus leavis mesp related genes in vertebrate cardiogenesis |
| title_sort | functional analysis of xenopus leavis mesp related genes in vertebrate cardiogenesis |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027235437&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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