Functions of microRNAs in the developing midbrain-hindbrain of the mouse:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20121005-1108535-0-9 Inhaltsverzeichnis |
| Item Description: | Zsfassung in dt. Sprache |
| Physical Description: | 159 Bl. Ill., graph. Darst. |
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Record in the Search Index
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| adam_text | IMAGE 1
CONTENT
1.0 ABSTRACT 1.1 ZUSAMMENFASSUNG 6
1.2 ABSTRACT 8
2.0 INTRODUCTION 2.1.1 BIOGENESIS OF MIRNAS AND MECHANISM OF ACTION 11
2.1.2 MIRNAS EXPRESSIONS AND FUNCTIONS IN THE DEVELOPING CNS 13
2.2 THE DEVELOPMENT OF CNS OF THE MOUSE 16
2.2.1 DEVELOPMENT OF THE VENTRAL-MID-/HINDBRAIN (VMH) NEURONAL
POPULATIONS OF THE MOUSE MIRNAS EXPRESSIONS AND FUNCTIONS IN THE
DEVELOPING CNS 16
2.2.2 GENETIC MECHANISMS INVOLVED IN DIFFERENTIATION OF MDDA NEURON 16
2.2.3 GENETIC MECHANISMS INVOLVED IN DEVELOPMENT OF OM/TN AND RN 20
2.3 AIMS OF THE PRESENT WORK 22
3.0 RESULT 3.1 MIRNAS THAT MIGHT BE INVOLVED IN THE REGULATION OF THE
MHB GRN AND MESDA NEURON DEVELOPMENT AS PREDICTED BY COMPUTATIONAL
ALGORITHMS 23 3.1.1 ESTABLISHMENT OF LNA-ISH DETECTION METHOD AND THE
VALIDATION OF
MIRNA EXPRESSION PATTERNS 28
3.1.1.1 MIRNAS THAT ARE EXPRESSED IN THE DEVELOPING MOUSE CNS 30
3A. .2MIR-144 IS SPECIFICALLY EXPRESSED IN THE LIVER DURING MOUSE
EMBRYONIC DEVELOPMENT 35
3.1.2FUNCTIONAL VALIDATION OF PREDICTED TARGET GENES FOR THE MIRNAS THAT
SHOWED EXPRESSION IN THE CNS DURING DEVELOPMENT 36
3.1.2.1 MIR-709 BUT NOT MIR-705 TARGETS WNTL 3 UTR 37
3.1.2.2 MIR-712 TARGETS WNT5B 3 UTR IN VITRO 39
3.1.2.3 MIR-714 TARGETS LEFL 3 UTR BUT NOT PITX3 3 UTR 41 3.1.2.4
MIR-135B DOES NOT TARGET OTX2- AND WNT6 3 UTR 42 3.1.3 A NEW PREDICTION
APPROACH TO PREDICT PUTATIVE MIRNAS THAT TARGET WNTL 3 UTR 43
3.1.4EXPERIMENTAL VALIDATION OF A PUTATIVE NOVEL MIRNA RECOGNITION
MOTIVE/TARGET ENHANCER SIGNAL 45
3.2 MIRNAS THAT MIGHT BE INVOLVED IN VMH NEURON DEVELOPMENT AS
DETERMINED BY MIRNA EXPRESSION PROFILING OF DIFFERENTIATING MESCS 3.2.1
DIRECTED DIFFERENTIATION OF MOUSE ESC INTO MDDA AND OTHER TYPES OF
VENTRAL MID/HINDBRAIN NEURONS 50
3.2.2NGS PROFILING OF MIRNAS EXPRESSED DURING THE DIRECTED
DIFFERENTIATION OF MOUSE ESCS INTO VMH NEURONS 52
3.2.3 EXPRESSION OF MIR-125B IN THE VENTRAL ANTERIOR NEURAL TUBE O F THE
MIDGESTATIONAL MOUSE EMBRYO 55
3.2.4EXPRESSION O F MIR-125B OVERLAPS WITH THE MOTONEURON MARKERS PHOX2B
AND SIML IN THE BASAL PLATE OF THE MURINE MID- AND HINDBRAIN 59
2
HTTP://D-NB.INFO/1034538470
IMAGE 2
3.2.5 MIR-125B DIRECTLY TARGETS THE PHOX2B AND SIML 3 UTRS 62
3.2.6 MIR-125B REGULATES THE DEVELOPMENT OF POSTMITOTIC PHOX2B + /ISLL +
OM/TN NEURONS AND O F NKX6-1 + BP PROGENITORS IN VITRO 66
3.2.7 THE NUMBERS OF PROLIFERATE CELLS IN PRIMARY VMH CELLS INCLUDING
NKX6-1 + BP PROGENITORS ARE DECREASED BY MIR-125B OVEREXPRESSION 75
3.2.8 REDUCTION OF THE MIR-125B DOSAGE IN VMH PRIMARY CULTURES ALSO
INCREASES THEIR SURVIVAL 77
3.3 GENERATION OF SHH-CRE-SBEL TRANSGENIC MICE FOR THE FATE-MAPPING AND
INACTIVATION O F CONDITIONAL ALLELES IN THE ROSTRAL MIDBRAIN/CAUDAL
DIENCEPHALON 79
4.0 DISCUSSION 4.1 EXPERIMENTAL VALIDATION O F BIOINFORMATICS PREDICTED
MIRNAS PREDICTION OF MIRNAS WITH BIOINFORMATICS TOOLS IS STILL NOT A
VERY RELIABLE METHOD TO DETECT NOVEL MIRNAS AND THEIR FUNCTIONAL
INTERACTIONS WITH TARGET
MRNAS 81
4.2 A CONSERVED PRIMATE MIRNA RECOGNITION MOTIF WAS NOT VALIDATED ON A
FUNCTIONAL LEVEL FOR THE MOUSE 88
4.3 4.3.1 MIR-125B IS EXPRESSED IN A VERY SPECIFIC SPATIOTEMPORAL
PATTERN IN THE DEVELOPING MURINE MHR THAT CORRELATES WITH THE EXPRESSION
OF ITS DIRECT TARGETS PHOX2B AND SIML 94
4.3.2 ANALYSES OF MIR-125B FUNCTION IN MOUSE VMH NEURAL DEVELOPMENT..
.96 4.4 OUTLOOK: MIR-125B FUNCTION IN VMH NEURON DEVELOPMENT 104
5.0 MATERIALS AND METHODS 5.1 MATERIALS 5.1.1 GENERAL EQUIPMENTS 105
5.1.2 CONSUMABLES 106
5.1.3 CHEMICALS 108
5.1.4 COMMERCIAL KITS 109
5.1.5 ENZYMES 109
5.1.6 ANTIBODIES 110
5.1.7BUFFERS AND SOLUTIONS 110
5.1.8 LNA OLIGO PROBE TO DETECT MIRNA EXPRESSION 113
5.1.9 RIBOPROBES TO DETECT MRNA 114
5.1.10 PRECURSOR MIRNAS FOR LUCIFERASE ASSAY 114
5.1.11 VECTORS 114
5.1.12 PRIMERS 115
5.1.12.1 GENOTYPING 115
5.1.12.2 LUCIFERERASE SENSOR VECTORS 115
5.1.12.3 SITE-DIRECTED MUTAGENESIS ON LUCIFERASE VECTOR 116 5.1.12.4
SITE-DIRECTED MUTAGENESIS FOR WNTL 3 UTR MIRNA RECOGNITION MOTIVE 116
5.1.12.5 MIRNA OVEREXPRESSING (OE) CONSTRUCT 117
5.1.12.6 LENTIVIRUS CONSTRUCTS 117
5.1.12.7 RT-QPCR TAQMAN PRIMERS 118
5.1.13 CELL CULTURE MEDIUM 118
3
IMAGE 3
5.2 METHODS
5.2.1 EXPERIMENTAL ANIMALS 120
5.2.2 GENOTYPING 5.2.2.1 ISOLATION OF GENOMIC DNA 120
5.2.2.2 PCR-GENOTYPING 120
5.2.2.3 R26R REPORTER MICE-GENOTYPING 121
5.2.3 HISTOLOGY 5.2.3.1 DISSECTION OF MOUSE EMBRYOS/BRAINS 121
5.2.3.2 PARAFFIN EMBEDDING O F MOUSE EMBRYOS 121
5.2.4 IN SITU HYBRIDIZATION (ISH) ON PARAFFIN SECTIONS 5.2.4.1 LABELLING
O F LOCKED NUCLEIC ACID (LNA) OLIGO DETECTION PROBE 122
5.2.4.1.1 DIG-LABELED LNA OLIGO DETECTION PROBE 122 5.2.4.1.2
RADIOACTIVE-LABELED LNA OLIGO DETECTION PROBE 122 5.2.4.2 RIBOPROBES FOR
RNA ISH 123
5.2.4.2.1 CLONING O F RIBOPROBES 123
5.2.4.2.2 LABELLING O F RIBOPROBES 123
5.2.4.3 PROCESSING O F PARAFFIN SECTION FOR LNA ISH AND RNA ISH 124
5.2.4.4DETECTION OF MIRNA ISH WITH LNA PROBE ON PARAFFIN SECTIONS 124
5.2.4.4.1 DIG-LABELLED LNA DETECTION PROBES 124
5.2.4.4.2 RADIOACTIVE-LABELLED LNA DETECTION PROBES 125 5.2.4.5
DETECTION O F MRNA ISH WITH RIBOPROBE ON PARAFFIN SECTIONS...125
5.2.4.6EXPOSURE AND DEVELOPING OF RADIOACTIVELY LABELLED SECTIONS....
126 5.2.5 IMMUNOHISTOCHEMISTRY 126
5.2.6 VECTOR CONSTRUCTS 126
5.2.6.1 LUCIFERASE SENSOR VECTORS 126
5.2.6.2 SITE-DIRECTED MUTAGENESIS ON LUCIFERASE VECTOR 127 5.2.6.3
SITE-DIRECTED MUTAGENESIS FOR WNTL 3 UTR MIRNA RECOGNITION MOTIF
EXPERIMENTS 127
5.2.6.4 MIRNA OVEREXPRESSION (OE) CONSTRUCT 127
5.2.6.5 LENTIVIRAL VECTORS GENERATION AND LENTIVIRUS PACKAGING 128 5.2.7
RESTRICTION ENZYME DIGESTION/BLUNTING/KINASING 128
5.2.8 LIGATIONS 128
5.2.9 TRANSFORMATION INTO COMPETENT BACTERIA 129
5.2.10 DNA MINI-AND MAXI-PREPS 129
5.2.11 SEQUENCING OF DNA 129
5.2.12 LUCIFERASE SENSOR ASSAY 129
5.2.13 MOUSE ES CELL DIFFERENTIATION 130
5.2.14 PRIMARY CELL CULTURE 5.2.14.1 COVER SLIP TREATMENT AND COATING
130
5.2.14.2 PRIMARY VMH CULTURE 131
5.2.14.3 EDU TREATMENT AND STAINING 131
5.2.14.4 IMMUNOCYTOCHEMISTRY 131
5.2.14.5 CELL COUNTING 132
5.2.15 ISOLATION OF TOTAL RNA 132
5.2.16 CDNA SYNTHESIS 132
5.2.17 QUANTITATIVE RT-PCR (QPCR) 132
4
IMAGE 4
5.2.18 MIRNA PROFILING WITH NEXT GENERATION SEQUENCING (NGS) 133
5.2.19 STATISTICAL ANALYSES 134
5.2.20 X-GAL HISTOCHEMISTRY 134
5.2.20.1 WHOLE MOUNT X-GAL STAINING 134
5.2.20.2 X-GAL STAINING ON CRYOSECTION 134
5.2.21 MICROSCOPY 134
6.0 REFERENCES 136
7.0 APPENDIX 7.1 ABBREVIATIONS 144
7.2 INDEX OF FIGURES AND TABLES 147
7.3 SUPPLEMENTARY FIGURES , 7.3.1 MIRN AS PREDICTED BY FIVE COMBINED
COMPUTATIONAL ALGORITHMS EXPRESSION ANALYSES 150
7.3.2NGS MIRNA PROFILING OF DIFFERENTIATING MESCS INTO VMH NEURONS -
VALIDATION OF THE PROFILING APPROACH 154
7.3.3 SEQUENCE OF THE SHH-CRE-SBEL CONSTRUCT USED FOR PRODUCTION OF
SHH-CRESBE1 TRANSGENIC MICE (CLONE A5) 155
7.4 ACKNOWLEDGMENT 157
7.5 CURRICULUM VITAE 158
5
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| spelling | Ng, Yen Kar Verfasser aut Functions of microRNAs in the developing midbrain-hindbrain of the mouse Yen Kar Ng 2012 159 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache München, Techn. Univ., Diss., 2012 Zelldifferenzierung (DE-588)4067536-1 gnd rswk-swf miRNS (DE-588)7736808-3 gnd rswk-swf Mittelhirn (DE-588)4074893-5 gnd rswk-swf Maus (DE-588)4169148-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Maus (DE-588)4169148-9 s Mittelhirn (DE-588)4074893-5 s miRNS (DE-588)7736808-3 s Zelldifferenzierung (DE-588)4067536-1 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20121005-1108535-0-9 http://mediatum.ub.tum.de/node?id=1108535 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20121005-1108535-0-9 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025778524&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ng, Yen Kar Functions of microRNAs in the developing midbrain-hindbrain of the mouse Zelldifferenzierung (DE-588)4067536-1 gnd miRNS (DE-588)7736808-3 gnd Mittelhirn (DE-588)4074893-5 gnd Maus (DE-588)4169148-9 gnd |
| subject_GND | (DE-588)4067536-1 (DE-588)7736808-3 (DE-588)4074893-5 (DE-588)4169148-9 (DE-588)4113937-9 |
| title | Functions of microRNAs in the developing midbrain-hindbrain of the mouse |
| title_auth | Functions of microRNAs in the developing midbrain-hindbrain of the mouse |
| title_exact_search | Functions of microRNAs in the developing midbrain-hindbrain of the mouse |
| title_full | Functions of microRNAs in the developing midbrain-hindbrain of the mouse Yen Kar Ng |
| title_fullStr | Functions of microRNAs in the developing midbrain-hindbrain of the mouse Yen Kar Ng |
| title_full_unstemmed | Functions of microRNAs in the developing midbrain-hindbrain of the mouse Yen Kar Ng |
| title_short | Functions of microRNAs in the developing midbrain-hindbrain of the mouse |
| title_sort | functions of micrornas in the developing midbrain hindbrain of the mouse |
| topic | Zelldifferenzierung (DE-588)4067536-1 gnd miRNS (DE-588)7736808-3 gnd Mittelhirn (DE-588)4074893-5 gnd Maus (DE-588)4169148-9 gnd |
| topic_facet | Zelldifferenzierung miRNS Mittelhirn Maus Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1108535 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20121005-1108535-0-9 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025778524&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT ngyenkar functionsofmicrornasinthedevelopingmidbrainhindbrainofthemouse |