Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells: transcriptome analysis of contractile SMCs
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2008
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 115 S. Ill., graph. Darst. 21 cm |
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| 245 | 1 | 0 | |a Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells |b transcriptome analysis of contractile SMCs |c vorgelegt von Shiva Rama Krishna Prasad Potta |
| 264 | 1 | |c 2008 | |
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Datensatz im Suchindex
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| adam_text | Titel: Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from em
Autor: Potta, Shiva Rama Krishna Prasad
Jahr: 2008
Table of Contents
Chap. Description__________^________________________________ Page(s)
Abbreviations 1 2
I. Introduction 3 14
1.1 Embryogenesis and Stem cell hierarchy
1.2 Signaling pathways and transcription factors involved in maintenance of Embryonic 4
7
Stem Cell (ESC) piuripotency and spontaneous differentiation in vitro
1.3 Embryonic Stem Cells as a developmental model In vitro
1A Smooth Muscle Cells Heterogeneity and Gene expression patterns 1 °
1.5 Embryonic Origins of Cardiac Cell Lineages
1.6 Aim of the work 14
II. Materials and Methods i5-40
1 Materials 15
1.1 Laboratory materials ^
1.2 Instruments and equipments ^°
1.3 Kits 16
1.4 Enzymes, antibodies, and antibiotics ^ ^
1.5 BAC clones and Bacterial strains 18
1.6 Eukaryoiic cell Lines 18
1.7 Buffers and other solutions 18
1.8 Media for £. co/i culture 19
1.9 Media for ES ceil culture, differentiation and freezing 19
1.10 Oligonucleotides 20
2 Cell Biological Methods 22
2.1 Culture conditions of murine ESCs 22
2.2 Passaging of ES cells 22
2.3 Freezing ES cells 22
2.4 Thawing ES cells 23
2.5 In vitro differentiation of ES cells 23
2.6 Differentiation of ES cells in 2D culture system 23
2.7 Differentiation of ES cells in 3D culture system 24
2.8 Preparation of Embryonic Cardiomyocytes 24
3 Molecular biological methods 25
3.1 Isolation of BAC DNA 25
3.2 Isolation of Plasmid DNA 25
3.3 Restriction Enzyme Digestion of DNA 25
3.4 Ligation of DNA fragments 26
3.5 Preparation of Chemically Competent E.coli cells 26
3.6 Transformation of chemically competent E.coli cells 26
3.7 Preparation of Glycerol stock of bacterial culture 26
3.8 DNA sequencing 27
3.9 DNA agarose gel electrophoresis 27
3.10 Recovery of DNA fragments from agarose gel 27
3.11 Recombination-mediated genetic engineering 28
3.11.1 Oiigonucleotide Design 28
3.11.2 PCR 28
3.11.3 Transformation of the BAC host with Red/ET expression plasmid 29
3.11.4 Subcloning of the gene of interest from BAC clone 30
3.12 Construction of the Targeting Vector 30
3.13 Construction of the Reporter plasmid with Acta2 promoter 31
3.14 Generation of the Stable Transgenic Acta2 ES cell line 32
3.15 Total RNA and cDNA preparation 33
3.16 Semiquantitatlve RT-PCR 34
3.17 Immunocytochemistry 34
3.18 Electrophysiological recordings 35
3.19 Contractility experiments 36
3.20 Flow cytometry 36
3.21 Microarray 37
3.22 Quantitative real-time PCR 39
III. Results 41
1 Generation, Characterization and Transcriptome Analysis of Contractile Smooth 41
Muscle Cells (SMCs)
1.1 Differentiation and Isolation of Contractile Acta2* SMCs from the transgenic ESCs 41
1.2 Characterization of the Acta2* SMCs by RT-PCR detection of SM-specific genes 43
1.3 Characterization of the Acta2* SMCs by Immunostaining of SM-specific cytoskeletal 44
proteins
1.4 Electrophysiological recording 45
1.5 Agonist-induced contraction of Acta2* SMCs 48
1.6 Microarray expression profiling and validation of the microarray data by quantitative 49
real- time PCR
1.7 Specific Gene Ontology Biological Process annotations of genes differentially 50
expressed in Acta2* SMCs
1.8 GO enrichment analysis of the upregulated genes in Acta2* SMCs 52
1.9 GO enrichment analysis of the downregulated genes in Acta2* SMCs 66
1.10 Gene expression pattern in Acta2* SMCs, aortic SMCs, undifferentiated ESCs and 72
15-days differentiated ESCs
1.11 Common and unique upregulated gene expression signatures between Acta2* and 74
aortic SMCs
1.12 Common and unique downregulated gene expression signatures between Acta2 and 77
aortic SMCs
1.13 Proliferative capacity of Acta2* SMCs 8
2 Generation and Functional Characterization of Cardiomyocytes a0
2.1 Differentiation and Isolation of the Acta2+Cardiomyocytes from the transgenic ESCs 80
2.2 Characterization of the Acta2* cells by RT-PCR gene expression 82
2.3 Characterization of the Acta2* Cardiomyocytes by Immunostaining 83
2.4 Electrophysiological Characterization 84
IV. Discussion 89
1.1 Differentiation and Isolation of contractile Acta2* SMCs from ES cells 89
1.2 Characterization of the Acta2* SMCs 89
1.3 GO enrichment analysis of the upregulated genes in Acta2+ SMCs 91
1.4 GO enrichment analysis of the downregulated genes in Acta2* SMCs 95
1.5 Gene expression pattern in Acta2+ SMCs, aortic SMCs, undifferentiated ESCs and 15- 96
days differentiated ESCs
1.6 Common and Unique upregulated gene expression signatures between Acta2* and 97
aortic SMCs
1.7 Common and Unique downregulated gene expression signatures between Acta2* and 98
aortic SMCs
1.8 Proliferative capacity of Acta2* SMCs 98
1.9 Differentiation and Isolation of Acta2* cardiomyocytes from ES cells 100
1.10 Characterization of the Acta2* cardiomyocytes 101
1.11 Concluding Remarks 102
IV. Future Perspectives 104
V. Summary 105
VI. References 107
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| dewey-ones | 616 - Diseases |
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| dewey-tens | 610 - Medicine and health |
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| spelling | Potta, Shiva Rama Krishna Prasad 1977- Verfasser (DE-588)139811621 aut Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs vorgelegt von Shiva Rama Krishna Prasad Potta 2008 115 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Köln, Univ., Diss., 2009 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=018984025&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Potta, Shiva Rama Krishna Prasad 1977- Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs |
| subject_GND | (DE-588)4113937-9 |
| title | Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs |
| title_auth | Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs |
| title_exact_search | Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs |
| title_full | Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs vorgelegt von Shiva Rama Krishna Prasad Potta |
| title_fullStr | Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs vorgelegt von Shiva Rama Krishna Prasad Potta |
| title_full_unstemmed | Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile SMCs vorgelegt von Shiva Rama Krishna Prasad Potta |
| title_short | Generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells |
| title_sort | generation and functional characterization of smooth muscle cells and cardiomyocytes derived from embryonic stem cells transcriptome analysis of contractile smcs |
| title_sub | transcriptome analysis of contractile SMCs |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=018984025&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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