Analyses of hematopoiesis and lineage commitment from ES and iPS cells [Elektronische Ressource] / vorgelegt von Katharina Seiler

Kseiler

Sujets

• Biologie

Informations

• Publié par: technische_universitat_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 14 Mo

Extrait

Analyses of hematopoiesis and lineage
commitment from ES and iPS cells

von der Fakultät III – Prozesswissenschaften
der Technischen Universität Berlin genehmigte
Dissertation

zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
- Dr. rer. nat. -


vorgelegt von Diplom-Biochemikerin
Katharina Seiler

durchgeführt am
Max-Planck-Institut für Infektionsbiologie, Berlin


Promotionsausschuss:
Vorsitzender: Prof. Dr. rer. nat. Ulf Stahl
Berichter: Prof. Dr. rer. nat. Fritz Melchers
Berichter: Prof. Dr. rer. nat. Roland Lauster
Tag der wissenschaftlichen Aussprache: 21.01.2011


Berlin, Januar 2011
D83



Meinen Eltern

























So eine Arbeit wird eigentlich nie fertig,
man muss sie für fertig erklären,
wenn man nach Zeit und Umständen
das mögliche getan hat. Zitat: Johann Wolfgang von Goethe
II Table of contents
Table of contents
Abbreviations......................................................................................................................... VII
1 Abstract ............................................................................................................................. 1
2 Zusammenfassung............................................................................................................ 3
3 Introduction...................................................................................................................... 6
3.1 Embyronic stem (ES) and induced pluripotent stem (iPS) cells .......................................... 6
3.1.1 Characteristics of mouse ES cells........................................................................................................6
3.1.2 Generating ES cell-like cells from somatic cells - the iPS cells .........................................................7
3.2 Hematopoiesis........................................................................................................................... 8
3.2.1 Early hematopoiesis.............................................................................................................................8
3.2.2 B and T lymphopoiesis........................................................................................................................9
3.2.3 The surrogate preB and preT cell receptor chains to follow B- and T-lymphopoiesis .....................12
3.3 Using ES and iPS cells to study hematopoiesis in vitro and for clinical application......... 14
3.3.1 In vitro generation of lymphoid, myeloid and erythroid cells from embryonic stem cells...............14
3.3.2 In vitro generation of HSCs from ES cells........................................................................................15
4 Thesis Objectives ............................................................................................................ 17
5 Materials and Methods................................................................................................... 19
5.1 Material................................................................................................................................... 19
5.1.1 Mice ...................................................................................................................................................19
5.1.2 Cell lines ............................................................................................................................................19
5.1.2.1 Eukaryotic cell lines ..............................................................................................................19
5.1.2.2 Bacterial cell lines .................................................................................................................20
5.1.3 Lab equipment ...................................................................................................................................20
5.1.4 Disposables........................................................................................................................................21
5.1.5 Chemicals and Supplements..............................................................................................................22
5.1.6 Buffers, Media and Additives............................................................................................................23
5.1.7 Antibodies and Avidin Conjugates....................................................................................................26
5.1.8 Oligonucleotides................................................................................................................................27
5.1.9 Enzymes.............................................................................................................................................28
5.1.10 Kits.....................................................................................................................................................29
5.1.11 Software.............................................................................................................................................29
5.1.12 Vectors...............................................................................................................................................29
5.2 Molecular methods................................................................................................................. 30
5.2.1 Growth and storage of bacteria..........................................................................................................30
5.2.2 Preparation of electrocompetent bacteria for transformation............................................................30
III Table of contents
5.2.3 Electrotransformation of competent bacteria ....................................................................................30
5.2.4 PCR....................................................................................................................................................31
5.2.5 Small scale (mini-prep) and large scale preparation (maxi-prep) of plasmid DNA .........................32
5.2.6 Restriction enzyme digestion of plasmid DNA or PCR-products.....................................................32
5.2.7 Gel electrophoresis ............................................................................................................................32
5.2.8 Gel extraction of DNA ......................................................................................................................33
5.2.9 Ligation..............................................................................................................................................33
5.2.10 BAC DNA purification......................................................................................................................33
5.2.11 Generation of the targeting construct ................................................................................................33
5.2.12 Preparation of the targeting cassette..................................................................................................34
5.2.13 BAC modification by homologous recombination ...........................................................................34
5.2.14 Verification of successfully modified BACs.....................................................................................35
5.2.15 Preparation of genomic DNA............................................................................................................35
5.2.16 Preparation of tail DNA and genotyping...........................................................................................36
5.2.17 Purification of RNA...........................................................................................................................36
5.2.18 RT-PCR .............................................................................................................................................36
5.2.19 Quantitative RT-PCR ........................................................................................................................36
5.3 Cellular work.......................................................................................................................... 37
5.3.1 Freezing and thawing of cultured cells..............................................................................................37
5.3.2 Determination of the cell number (counting) ....................................................................................37
5.3.3 Maintenance of OP9 and OP9-DL1 stromal cells .............................................................................37
5.3.4 Maintenance of ST2 stromal cells .....................................................................................................38
5.3.5 Preparation of embryonic fibroblasts (EF cells)................................................................................38
5.3.6 Production of growth factor conditioned hybridoma cell culture supernatants ................................39
5.3.7 Maintenance of embryonic stem (ES) cells.......................................................................................39