Analysis of the expression pattern and knock-out phenotype of Slit-like 2 (Slitl2) in the mouse [Elektronische Ressource] / vorgelegt von Anja Michaela Mayer

Anja Michaela Mayer

Sujets

• Biologie

Informations

• Publié par: freie_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 58
• Langue: Deutsch
• Poids de l'ouvrage: 13 Mo

Extrait

Aus dem
Institut für Tierpathologie
des Fachbereichs Veterinärmedizin
der Freien Universität Berlin
und dem
Max-Planck-Institut für molekulare Genetik
Analysis of the expression pattern
and knock-out phenotype of
Slit-like 2 (Slitl2) in the mouse
Inaugural-Dissertation
zur Erlangung des Grades eines
Doktors der Veterinärmedizin
an der
Freien Universität Berlin
vorgelegt von
Anja Michaela Mayer
Tierärztin aus
Mühldorf am Inn
Berlin 2009
Journal-Nr.: 3359Gedruckt mit Genehmigung
des Fachbereichs Veterinärmedizin
der Freien Universität Berlin
Dekan: Univ.-Prof. Dr. med. vet. Leo Brunnberg
Erster Gutachter: Univ.-Prof. Dr. med. vet. Achim D. Gruber, Ph.D.
Zweiter Gutachter: Univ.-Prof. Dr. Bernhard G. Herrmann
Dritter Gutachter: Univ.-Prof. Dr. Michael Schmidt
Deskriptoren (nach CAB-Thesaurus): mice, laboratory animals, gene expression,
genes, reporter [MeSH], membrane proteins, gene expression profiling [MeSH],
mice, knockout [MeSH]
Tag der Promotion: 19.02.2010Für meine Mutter,
durch die ich werden konnte, wer ich bin.
Und für Peter,
bei dem ich sein kann, wer ich bin.i
Contents
Abbreviations iii
1 Introduction 1
1.1 Development of the mouse embryo . . . . . . . . . . . . . . . . . 1
1.2 Devt of the kidney . . . . . . . . . . . . . . . . . . 3
1.3 Glomeruli at a glance . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 The Slit genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.5 The Slit-like 2 gene . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.6 TGF-1 in development and disease . . . . . . . . . . . . . . . . . 11
1.7 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2 Materials and methods 15
2.1 Mouse strains and animal husbandry . . . . . . . . . . . . . . . . 15
2.2 Generation of Slitl2-mutant mouse lines . . . . . . . . . . . . . . 16
2.2.1 Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2.2 Generation of ES cell lines . . . . . . . . . . . . . . . . . . 23
2.2.3 Screening of ES cell clones . . . . . . . . . . . . . . . . . . 25
2.2.4 Generation of mouse lines . . . . . . . . . . . . . . . . . . 29
2.2.5 Genotyping of mice . . . . . . . . . . . . . . . . . . . . . . 29
2.2.6 Generation of mouse primary embryonic fibroblasts . . . . 32
2.3 Molecular and cellular biology . . . . . . . . . . . . . . . . . . . . 33
2.3.1 Northern blot analysis . . . . . . . . . . . . . . . . . . . . 33
2.3.2 cDNA microarray analysis . . . . . . . . . . . . . . . . . . 34
2.3.3 Quantitative real-time PCR . . . . . . . . . . . . . . . . . 36
2.3.4 Western blot analysis . . . . . . . . . . . . . . . . . . . . . 38
2.3.5 Flow cytometric analysis . . . . . . . . . . . . . . . . . . . 40
2.4 Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.4.1 Standard staining procedures . . . . . . . . . . . . . . . . 42
2.4.2 Whole-mount in situ hybridization . . . . . . . . . . . . . 44
2.4.3 X-gal staining . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.4.4 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . 49
2.4.5 Immunocytoc . . . . . . . . . . . . . . . . . . . . 51
2.4.6 Electron microscopy . . . . . . . . . . . . . . . . . . . . . 51
2.4.7 Skeleton staining . . . . . . . . . . . . . . . . . . . . . . . 52
2.4.8 Micro-computed tomography . . . . . . . . . . . . . . . . . 52
2.5 Evaluation of clinical laboratory parameters . . . . . . . . . . . . 52
2.5.1 Blood parameters . . . . . . . . . . . . . . . . . . . . . . . 52
2.5.2 Urinary parameters . . . . . . . . . . . . . . . . . . . . . . 54ii
2.6 General buffers, solutions, and chemicals . . . . . . . . . . . . . . 56
3 Results 59
3.1 Slitl2 expression pattern . . . . . . . . . . . . . . . . . . . . . . . 59
3.1.1 Endogenous Slitl2 expression . . . . . . . . . . . . . . . . 59
3.1.2 Slitl2-LacZ expression . . . . . . . . . . . . . . . . . . . . 64
3.1.3 Slitl2-Venus . . . . . . . . . . . . . . . . . . . . 74
3.2 Generation of Slitl2-mutant mice . . . . . . . . . . . . . . . . . . 78
3.2.1 Gene targeting of the mouse Slitl2 locus . . . . . . . . . . 78
3.2.2 Generation of Slitl2-floxed-neo mice . . . . . . . . . . . . . 80
3.2.3 of conditional Slitl2-floxed mice . . . . . . . . . 81
3.2.4 Generation of Slitl2-null mice . . . . . . . . . . . . . . . . 83
3.3 Phenotypic analysis of Slitl2-deficient mice . . . . . . . . . . . . . 84
3.3.1 General characteristics . . . . . . . . . . . . . . . . . . . . 84
3.3.2 Kidney phenotype of Slitl2-deficient mice . . . . . . . . . . 87
3.3.3 Bone phenotype of Slitl2t mice . . . . . . . . . . . 98
3.3.4 Thymus and spleen phenotype of Slitl2-deficient mice . . . 102
3.4 Gene expression profiling . . . . . . . . . . . . . . . . . . . . . . . 109
3.4.1 cDNA microarray analysis . . . . . . . . . . . . . . . . . . 109
3.4.2 Quantitative real-time PCR . . . . . . . . . . . . . . . . . 113
4 Discussion 115
5 Summary 125
6 Zusammenfassung 127
References 129
List of Figures 141
List of Tables 143
Appendices 145
Presentations arising from this thesis 155
Acknowledgments 157
Candidate’s declaration 159iii
Abbreviations
- anti-
aa amino acid(s)
AP alkaline phosphatase
BAC bacterial artificial chromosome
bp base pair(s)
BSA bovine serum albumin
Cam chloramphenicol
CD cluster of differentiation
cDNA complementary DNA
CMV cytomegalovirus
cRNA complementary RNA
DAPI 4’,6-diamidino-2-phenylindole
ddH O double-distilled H O2 2
dig digoxigenin
E embryonic day
ES embryonic stem
EtOH ethanol
FBS fetal bovine serum
FCS fetal calf serum
g gravity
het heterozygous
hom homozygous
GBM glomerular basement membrane
GFB filtration barrier
GFP green fluorescent protein
h hour(s)
H&E hematoxylin and eosin
HEK human embryonic kidney
Ig immunoglobulin
IQR interquartile range
Kan kanamycin
kb kilobaseiv
kDa kilodalton
KO knock-out
LB Luria Bertani
CT micro-computed tomography
MEFs mouse primary embryonic fibroblasts
MetOH methanol
MHC major histocompatibility complex
min minute(s)
neo neomycin
OD optical density
o/n overnight
ORF open reading frame
P postnatal day
PAS periodic acid-Schiff
PCR polymerase chain reaction
PBS phosphate-buffered saline
RBC red blood cell(s)
rRNA ribosomal RNA
RT room temperature
RT-PCR reverse transcription PCR
SD standard deviation
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
sec second(s)
TCR T-cell receptor
UTR untranslated region
vol. volume
VSMCs vascular smooth muscle cells
v/v volume/volume
WBC white blood cell(s)
WISH whole-mount in situ hybridization
wt wild-type
w/v weight/volume
YFP yellow fluorescent protein1
1 Introduction
1.1 Development of the mouse embryo
Mouse embryonic development takes 19 days on average. It begins with a single
cell, namely the fertilized egg, or zygote. The zygote undergoes cleavage divisions
to form blastomeres. At the 8-cell stage, the embryo has developed into a morula,
and its cells start to compact, polarize, and form a blastocyst. Implantation of
the blastocyst occurs around embryonic day 4.5 (E4.5), and by this stage, two
distinct cell populations can be discriminated: the outer trophoblast cells and
the inner cell mass (ICM). The latter comprises two cell layers, namely the hy-
poblast, also known as the primitive endoderm, and the epiblast. The embryo
proper is formed exclusively from descendants of the epiblast. At the onset of
the gastrulation process, the epiblast consists of approximately 800 cells [Snow
and Bennett, 1978]. Gastrulation in the mouse embryo begins around E6.5 when
the primitive streak at the future posterior end of the embryo arises. Cells from
the continuous epiblast layer detach from their neighboring cells by a process
known as epithelial-to-mesenchymal transition. Thus, the cells become motile
and individually migrate through the streak [Burdsal et al., 1993]. One part of
the ingressing cells constitutes an intermediate layer of embryonic mesoderm be-
tween the epiblast and the underlying hypoblast, while the other part gradually
replaces the hypoblast cells, thereby forming the embryonic endoderm. As a re-
sult of gastrulation, the embryo becomes a trilaminar entity, with an outer layer
of ectoderm, an intermediate mesoderm, and an inner layer of endoderm. In the
mouse, the germ layers are initially arranged in an inverse manner with the ecto-
derm facing the inside and the endoderm facing the outer aspect of the U-shaped
egg cylinder. Around E8.5, this inversion is reverted by the complex process of
turning. The onset of organogenesis coincides with turning. During this phase,
the germ layers begin to assemble the organ and tissue rudiments. Also, several
transient structures emerge, e.g., the notochord, the branchial arches, and the
somites. They ultimately contribute to the generation of functional organs or
tissues in the adult. Distinct cell types derive from the different germ layers. The