Epigenetic reprogramming in mouse germ cells [Elektronische Ressource] / von Petra Hajkova

Peter Williamson

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2002
• Nombre de lectures: 39
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Epigenetic reprogramming in mouse germ cells




D i s s e r t a t i o n


zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
( Dr. rer. nat.)
im Fach Biologie
eingereicht an der


Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von

Mgr. Petra Hajkova
geboren am 5. November 1973 in Jesenik



Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jürgen Mlynek

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Michael Linscheid

Gutachter/innen: 1. Prof. Dr. Jörn E. Walter
2. Prof. Dr. Harald Saumweber
3. Dr. Wolf Reik
Tag der mündlichen Prüfung: 16. September 2002










to my parents























2Erklärung


Hiermit erkläre ich, daß ich die vorliegende Arbeit selbständig und ohne Benutzung
anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen
oder indirekt übernommenen Daten und Konzepte sind uter Angabe der Quelle
gekennzeichnet.

Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher
Form in anderen Prüfunsverfahren vorgelegt.



































3TABLE OF CONTENTS

1 INTRODUCTION_________________________________________________9
1.1 Brief history of DNA methylation............................................................................................9
1.2 Genomic imprinting and its connection to DNA methylation ................................................11
1.3 The “life cycle” of imprinting in the mammalian development .............................................17
1.4 Imprinting and methylation changes during early embryogenesis .........................................18
1.5 Germ line and its key role in the epigenetic “life cycle” ........................................................20
1.6 Biological studies of primordial germ cells – origin, characteristics, development ...............21
1.6.1 Origin of primordial germ cells ......................................................................................21
1.6.2 Germ cells migration and colonisation of the embryonic genital ridges.........................23
1.6.3 Gametogenesis inside the developing gonads.................................................................25
1.6.4 Brief summary of spermatogenesis.................................................................................25
1.6.5 ummary of oogenesis ...........................................................................................26
1.6.6 Germ cell development is connected with epigenetic reprogramming ...........................28
1.6.7 Re-establishment of genomic imprints ...........................................................................29
1.6.8 Factors possibly involved in the establishment of gametic imprinting30
1.7 Molecular techniques used for DNA methylation studies ......................................................31
1.7.1 Non-specific methylation analysis..................................................................................31
1.7.2 Sequence-specific methylation analysis .........................................................................32
1.7.3 Bisulphite genomic sequencing......................................................................................33
1.8 The aim of the thesis...............................................................................................................35
2 MATERIAL & METHODS_________________________________________36
2.1 Material .................................................................................................................................36
2.1.1 Bacterial strains ..............................................................................................................36
2.1.2 Mouse strains..................................................................................................................36
2.1.3 Cloning vectors...............................................................................................................37
2.1.4 Enzymes.........................................................................................................................37
2.1.5 Chemicals & other material ............................................................................................38
2.1.5.1 Chemicals..................................................................................................................................38
2.1.5.2 DNA-molecular weight markers................................................................................................39
2.1.5.3 Kits............................................................................................................................................39
2.1.5.4 Other material...........................................................................................................................40
2.1.5.5 Equipment...................40
2.1.5.6 Bacterial media.................41
2.1.5.7 Buffers & solutions....................................................................................................................41
2.1.5.8 Media and chemicals for the germ cell preparation..................................................................42
2.1.6 Oligonucleotides....43
2.1.6.1 Primers for the sex determination of early mouse embryos ......................................................43
2.1.6.2 Primers for the colony PCR ......................................................................................................43
2.1.6.3 Sequencing primers...................................................................................................................43
2.1.6.4 Primers for the bisulphite analysis............................................................................................43
2.2 Methods .................................................................................................................................46
2.2.1 Isolation of mouse primordial germ cells .......................................................................46
2.2.1.1 Antibody based purification ......................................................................................................47
2.2.1.2 GFP-based purification ............................................................................................................48
2.2.2 Alkaline Phosphatase Staining .......................................................................................48
2.2.3 Sex determination of embryos for the isolation of primordial germ cells ......................48
2.2.4 Isolation of chromosomal DNA from tissue samples .....................................................49
2.2.5 Bisulphite based genomic sequencing ............................................................................49
2.2.5.1 The bisulphite treatment of single cell suspension ....................................................................50
2.2.5.2 Bisulphite treatment of isolated chromosomal DNA .................................................................51
2.2.6 Bisulphite PCR amplification .........................................................................................51
2.2.7 Isolation of a DNA fragment from the agarose gel.........................................................57
2.2.8 Cloning ...........................................................................................................................57
2.2.9 Preparation of competent E.coli Sure cells.....................................................................58
2.2.10 Detection of positive clones by colony PCR ..................................................................59
2.2.11 Sequencing and the sequence evaluation........................................................................60
2.2.12 mC immunofluorescent staining.....................................................................................61
43 RESULTS________________________________________________________62
3.1 Purification of primordial germ cells..................................................................................62
3.2 Methylation analysis using the bisulphite genomic sequencing........................................65
3.3 Methylation status of imprinted genes................................................................................66
3.3.1 Methylation changes in lit1 CpG island......................................................................67
3.3.2 Methylation changes in peg3 gene ...............................................................................69
3.3.3 Methylation changes in Igf2 gene71
3.3.4 Methylation changes in H19 gene75
3.3.5 Methylation changes in Snrpn gene..................................................