Cloning in cattle [Elektronische Ressource] : nuclear architecture and epigenetic status of chromatin during reprogramming of donor cell nuclei / Daniela Köhler

Daniela Koehler

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

Department Biologie II
Anthropologie und Humangenetik
Ludwig-Maximilians-Universität München
_____________________________

Cloning in Cattle:
Nuclear architecture and
epigenetic status of chromatin
during reprogramming of donor
cell nuclei
_____________________________



Daniela Köhler





Dissertation an der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
Eingereicht am 23.10.2008













Cloning in cattle: Nuclear architecture and
epigenetic status of chromatin during
reprogramming of donor cell nuclei

Dissertation an der Fakultät für Biologie, Department II
der Ludwig-Maximilians-Universität München (LMU)

Vorgelegt von
Dipl.Biol. Daniela Köhler

Gutachter:
Erstgutachter: Prof. Dr. Thomas Cremer
Zweitgutachter: Prof. Dr. Manfred Schliwa

Tag der mündlichen Prüfung: 19.März 2009












Einmal kündigte der Physiker Leo Szilard seinem Freund Hans Bethe an, er wolle
eventuell ein Tagebuch führen: “ Ich habe nicht vor etwas zu veröffentlichen. Ich
möchte die Tatsachen nur festhalten, damit Gott Bescheid weiß.” Daraufhin fragte
Bethe: “Glauben sie nicht, dass Gott die Tatsachen schon kennt?” – “Ja”, erwiderte
Szilard, “die Tatsachen kennt er. Aber diese Version der Tatsachen kennt er noch
nicht.”
Hans Christian von Baeyer, Das Atom der Falle
____________________________________________________________Contents

Contents:
1. Summary ______________________________________________________________1
1.1. Abstract_________________________________ 1
1.2. Zusammenfassung_________________________ 3
2. Introduction: Positions of chromosome territories (CTs) and sub-domains in the cell
nucleus___5
2.1. Goals of the study ________________________________ 5
2.2. Organization in a cell nucleus_______________ 7
2.2.1. Discovery of CTs_________________________ 7
2.2.2. The Chromosome-Territory-Interchromatin-Compartment (CT-IC) model________ 9
2.2.2.1. Intermingling and loops________________ 11
2.2.2.2. Computer-based models ________________________________ 12
2.2.3. Different compartments within a nucleus___ 12
2.2.3.1. Chromatin structure___________________ 13
2.2.3.2. Euchromatin and Heterochromatin______ 13
2.2.3.3. Nuclear lamina________________________ 15
2.2.3.4. Nucleolus, Speckles and other nuclear bodies_______________________________ 16
2.2.4. Distribution pattern of CTs: radial distribution and neighborhoods _____________ 16
2.2.4.1. Prometaphase rosettes _________________________________________________ 17
2.2.4.2. Interphase nuclei______________________ 17
2.2.4.3. Sperm heads__________________________ 18
2.2.4.4. Tissues and cancer_____________________ 18
2.2.4.5. Gene-rich and gene-poor chromosomes ___________________________________ 19
2.2.4.6. Differentiation and development_________ 20
2.2.4.7. Chromatin mobility & gene expression____ 21
2.2.5. Evolutionary conserved pattern___________ 23
2.2.6. Influence of nuclear shape on CT distribution _______________________________ 24
2.2.7. Preimplantation embryos as a model system_________________________________ 25
2.2.7.1. Diagnostics in preimplantation embryos___ 25
3. Material & Methods: ____________________________________________________27
3.1. Cell and embryo material__________________ 27
3.1.1. Human Diploid Fibroblasts (HDF)_________ 27
3.1.2. Lymphocytes (human/bovine)_____________ 27
3.1.3. Bovine Fetal Fibroblasts (BFF) ____________________________________________ 28
3.1.4. Bovine embryos_________________________ 28
3.1.5. Mouse embryos 28
3.2. Cell culture______________________________________________________________ 29
3.2.1. Media_________________________________ 29
I ____________________________________________________________Contents

3.2.2. Unfreezing, culture and freezing down______________________________________ 29
3.2.3. Growing cells on glass coverslips__________ 29
3.3. Generation of embryonic specimen 30
3.3.1. Extraction of oocytes ____________________________________________________ 30
3.3.2. In-vitro fertilization (IVF)________________ 31
3.3.3. Nuclear Transfer (NT)___________________ 32
3.4. Metaphase preparation____________________ 33
3.4.1. Treatment of cells_______________________ 33
3.4.2. Drop cells______________________________________________________________ 34
3.4.3. Post-treatment for conservation ___________________________________________ 34
3.4.4. Pepsin-treatment of slides________________ 35
3.5. Fixation of cells for 3D fluorescence in situ hybridization (FISH) _________________ 35
3.6. Fixation of embryos_______________________ 36
3.7. Probe amplification and labeling____________ 39
3.7.1. Origin of bovine probes __________________________________________________ 39
3.7.2. Nucleotide labeling______________________ 39
3.7.3. Secondary DOP-PCR____________________ 41
3.7.4. Whole Genome Amplification (WGA) by Multiple Displacement Amplification
(MDA) ________________________________________________________________ 42
3.7.5. Label DOP-PCR________________________ 43
3.7.6. Nick translation (NT)____________________ 44
3.7.7. Preparation of C t-1 DNA ________________________________________________ 45 0
3.8. Probe precipitation_______________________ 47
3.9. 2D FISH ________________________________________________________________ 47
3.9.1. 2D FISH set up_________________________ 47
3.9.2. 2D FISH detection______________________ 48
3.10. 3D FISH_______________________________ 49
3.10.1. 3D FISH on cells _______________________________________________________ 49
3.10.1.1. 3D FISH set up on cells________________ 49
3.10.1.2. Detection of 3D FISH on cells___________ 50
3.10.2. 3D FISH on embryos___________________ 50
3.10.2.1. 3D FISH set up on embryos ____________________________________________ 50
3.10.2.2. Detection of FISH signals on embryos____ 51
3.11. Microscopy _____________________________________________________________ 53
3.11.1. Stereomicroscope______________________ 53
3.11.2. Light microscope 53
3.11.3. Fluorescent microscope_________________ 53
3.11.4. Confocal laser scanning microscope (CLSM) _______________________________ 54
II ____________________________________________________________Contents

3.12. Data evaluation _________________________________________________________ 55
3.12.1. Shift correction________________________ 55
3.12.2. Deconvolution 55
3.12.3. Image J______________________________ 56
3.12.4. Quantitative data evaluation _____________________________________________ 57
3.12.4.1. Three dimensional – Relative Radial Distribution (3D-RRD)_________________ 58
3.12.4.2. Absolute Distance to Surface (ADS) or Enhanced Absolute Distance to Surface
(eADS)________________________________ 58
3.12.5. Photoshop (CS2, Adobe systems, Inc., S. Jose) ______________________________ 59
3.12.6. Amira________________________________ 59
3.12.7. Statistics 59
4. Results:_______________________________60
4.1. Similarity of chromosome arrangement is lost after two cell cycles in HeLa and normal
diploid cells______________________________________________________________ 60
4.2. Distribution of gene-rich and gene-poor CTs in dependency on nuclear shape and cell
cycle___________________________________ 61
4.2.1. Human fibroblasts data with flat shaped nuclei 61
4.2.1.1. Cell cycle dependent distribution of big vs. small chromosomes (HSA 1 vs. HSA 20) 61
4.2.1.2. Cell cycle dependent distribution of gene-rich vs. gene-poor chromosomes (HSA 17
vs. HSA Y) ___________________________________________________________ 63
4.2.1.3. Cell cycle dependent distribution of gene-poor vs. gene-rich chromosomes (HSA 18
vs. HSA 19) 64
4.2.2. Inducing a change of nuclear shape by growing fibroblasts on micropattern ______ 67
4.2.2.1. Morphology and shape of nuclei from fibroblasts grown on micropattern _______ 68
4.2.2.2. Distribution of human chromosomes 18 and 19 in human fibroblast cells 70
4.2.2.2.1. Cigar shaped cells____________________________________________________ 70
4.2.2.2.2. Round shaped cells___________________ 72
4.2.2.3. Distribution of human orthologous chromosomes 18 and 19 in fibroblasts of Wolf´s
guenon______________________________ 73
4.2.2.3.1. Cigar shaped cells____________________ 74
4.2.2.3.2. Round shaped cells___________________ 75
4.2.2.4. Distribution of bovine chromosomes 19 and 20 in fibroblast nuclei of Cattle _____ 76
4.2.2.4.1. Bovine fetal fibroblasts (BFF451-1): “normal” flat shape 77
4.2.2.4.2. Cigar shaped bovine fetal fibroblasts (BFF451-1)__________________________ 77
4.2.2.4.3. Round shaped bovine fetal fibroblasts (BFF 451-1) ________________________ 78
4.3. FISH on 3D preserved bovine preimplantation embryos 80
4.3.1. Establishment of a FISH protocol on 3D preserved embryos ___________________ 80
4.3.2. Embryo attachment ____________________