Endoderm lineage labelling using BAC reporter constructs in ES-cells and mouse embryos [Elektronische Ressource] / Sascha Imhof

Sascha Steinhoff

Sujets

• Naturwissenschaften
• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 35
• Langue: English
• Poids de l'ouvrage: 11 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Entwicklungsgenetik



Endoderm lineage labelling using
BAC reporter constructs
in ES cells and mouse embryos


SASCHA IMHOF



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. S. Scherer
Prüfer der Dissertation: 1. apl. Prof. Dr. J. Graw
2. Univ.-Prof. A. Schnieke, Ph.D.



Die Dissertation wurde am 11.05.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 31.07.2009 angenommen.





















Strength does not come
from physical capacity.
It comes from an
indomitable will.

Mahatma Gandhi
Indian political and spiritual
leader (1869 – 1948) Table of Contents

TABLE OF CONTENTS......................................................................... 1
1 INTRODUCTION ............................................................................. 7
1.1 The early development of the mouse embryo with a focus on
endoderm development ................................................................................ 7
1.1.1 The BMP signalling pathway .................................................................. 18
1.1.2 The transcription factor Foxa2: essential for endoderm
development ........................................................................................... 19
1.1.3 The transcription factor Sox17: necessary for proper endoderm
development ........................................................................................... 21
1.1.4 Brachyury (T): important for development of the mesoderm ................... 22
1.1.5 The role of Hhex during endoderm development and
organogenesis ........................................................................................ 23
1.1.6 The role of Nkx2.1 for endodermal organ development .......................... 25
1.1.7 The role of Pdx1 for endodermal organ development ............................. 26
1.2 BAC transgenes: A technology which allows expression of
reporter genes ............................................................................................. 28
2 SCOPE OF THE WORK ................................................................ 31
3 RESULTS ...................................................................................... 32
3.1 Generation of BAC transgenic embryonic stem (ES) cells and
mouse lines ................................................................................................. 32
3.2 Generation of a Hhex::Lyn-Tomato BAC transgene ................................. 34
3.2.1 Generation of Hhex BAC transgenic reporter ES cells ........................... 36
3.2.2 Integrity check of BAC transgenic ES cells by BAC end PCR ................ 38
3.2.3 In vitro expression of Hhex BAC transgenic ES cells in ES cells
and in vitro differentiated ES cells into endoderm ................................... 39
1 Table of Contents

3.2.4 In vivo analysis of Hhex::Lyn-Tomato BAC transgenic ES cells
by tetraploid complementation technique ............................................... 40
3.2.5 Expression analysis of the BAC transgenic Hhex::Lyn-Tomato
mouse line .............................................................................................. 43
3.3 Comparison of Hhex::Lyn-Tomato BAC transgenic mouse line
with a Hhex-GFP transgene mouse line .................................................... 47
3.4 Generation of a Sox17::H2B-Tomato BAC transgenic mouse line ......... 48
3.4.1 Generation of Sox17 BAC transgenic reporter ES cells .......................... 50
3.4.2 Integrity check of Sox17 BAC transgenic ES cells by BAC end
PCR ........................................................................................................ 51
3.4.3 In vitro expression of Sox::H2B-Tomato BAC transgenic ES cells
in in vitro differentiated ES cells into endoderm ...................................... 52
3.4.4 Analysis of a Sox17::H2B-Tomato BAC transgenic mouse line .............. 54
3.5 Generation of Foxa2::Lyn-Venus BAC transgenic ES cells and
expression analysis in tetraploid-derived mouse embryos .................... 55
3.5.1 Generation of Foxa2 BAC transgenic reporter ES cells ......................... 57
3.5.2 Integrity check of Foxa2 BAC transgenic ES cells by BAC end
PCR ........................................................................................................ 58
3.5.3 In vitro expression of Foxa2 BAC transgenic ES cells in in vitro
differentiated ES cells into endoderm ..................................................... 59
3.5.4 In vivo analysis of Foxa2::Lyn-Venus BAC transgenic ES cells by
tetraploid complementation technique .................................................... 59
3.6 Generation of Nkx2.1::H2B-Venus BAC transgenic ES cells and
expression analysis in tetraploid-derived mouse embryos .................... 62
3.6.1 Generation of Nkx2.1 BAC transgenic reporter ES cells ......................... 64
3.6.2 Integrity check of Nkx2.1 BAC transgenic ES cells by BAC end
PCR ........................................................................................................ 65
3.6.3 In vivo analysis of Nkx2.1::H2B-Venus BAC transgenic ES cells
by tetraploid complementation technique ............................................... 66
2 Table of Contents

3.7 Generation of a Pdx1::H2B-Venus BAC .................................................... 67
3.8 Whole mount Foxa2 antibody staining on Hhex::Lyn-Tomato
transgenic mouse embryos ........................................................................ 69
3.9 Whole mount embryo stainings using antibodies against active
Smad1/5/8 and Foxa2 .................................................................................. 72
3.10 Whole mount embryo stainings using antibodies against active
Smad1/5/8 and Brachyury (T) ..................................................................... 77
4 DISCUSSION................................................................................. 80
4.1 The efficiency of BAC transgene technology by bacterial
recombineering and the following analysis of BAC transgenic
ES cells in vitro and in vivo demonstrates advantages of BAC
transgenes ................................................................................................... 80
4.2 A Hhex::Lyn-Tomato BAC transgenic mouse line reflects the
accurate reporter expression and shows some new aspects of
Hhex expression in the mouse .................................................................. 86
4.3 The Hhex BAC transgenic mouse line shows a homogenous and
detailed expression pattern in comparison to a Hhex-GFP
transgenic line ............................................................................................. 87
4.4 A Sox17 BAC transgenic mouse line showns restricted
transgene expression in the definitive endoderm .................................... 88
4.5 Whole mount Foxa2 staining on Hhex::Lyn Tomato BAC
transgenic mouse embryos enable the mapping of populations
on cellular level ........................................................................................... 89
4.6 Whole mount Foxa2, T and active Smad1/5/8 staining in
gastrulation stage embryos ....................................................................... 90
5 MATERIALS .................................................................................. 94
5.1 Instruments .................................................................................................. 94
5.2 Chemicals .................................................................................................... 96
3 Table of Contents

5.3 Kits ............................................................................................................... 96
5.4 Commonly used stock solutions ............................................................... 96
5.5 Solutions for the work with bacteria .......................................................... 97
5.6 Solutions for cell culture ............................................................................ 98
5.7 Solutions for embryo work ......................................................................... 99
5.8 Solutions for Southern blot analysis ....................................................... 100
5.9 Solutions for immunochemistry .............................................................. 101
5.10 Enzymes ..................................................................................................... 101
5.11 Antibodies ..............................................................