Consequences of postnatal insulin-like growth factor II overexpression in insulin-like growth factor I deficient mice [Elektronische Ressource] / by Corinna Mörth

Corinna Mörth

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Institute of Molecular Animal Breeding and Biotechnology, Gene Center
Faculty of Veterinary Medicine of the Ludwig Maximilian University, Munich
Prof. Dr. Eckhard Wolf







Consequences of postnatal insulin-like growth factor II
overexpression in insulin-like growth factor I
deficient mice







Thesis for the attainment of the title Doctor in Veterinary Medicine
from the Faculty of Veterinary Medicine of the Ludwig Maximilian University, Munich



by
Corinna Mörth
from Munich, Germany

Munich, 2005



Aus dem Institut für Tierzucht der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München
Lehrstuhl für Molekulare Tierzucht und Biotechnologie, Genzentrum
Univ.-Prof. Dr. Eckhard Wolf







Konsequenzen dauerhaft erhöhter Spiegel des Insulin-
ähnlichen Wachstumsfaktors II in Insulin-ähnlichen
Wachstumsfaktor I defizienten Mäusen





Inaugural-Dissertation
zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München




von
Corinna Mörth
aus München

München 2005





Gedruckt mit der Genehmigung der Tierärztlichen Fakultät der

Ludwig-Maximilians-Universität München














Dekan: Univ.-Prof. Dr. A. Stolle

Referent: Univ.-Prof. Dr. E. Wolf

Korreferent: Univ. Prof. Dr. K. Pfister














Tag der Promotion 15. Juli 2005













MY FAMILY I Contents
__________________________________________________________________________________________
Contents
Page

Contes I

Abreviatons III

1 Introduction and objectives 1

2 Review of the literature 3
2.1 The insulin-like growth factor (IGF) system 3
2.1.1 Overview
2.1.2 IGF-I 6
2.1.2.1 gene
2.1.2.2 protein 6
2.1.2.3 Regulation of IGF-I expression
2.1.2.3.1 RNA 6
2.1.2.3.2 IGF-I levels in the circulation 8
2.1.2.4 Biological effects of IGF-I 9
2.1.2.4.1 Overview
2.1.2.4.2 Growth and development 11
2.1.2.4.3 Bone metabolism 11
2.1.2.4.4 Cancer 13
2.1.3 IGF-II
2.1.3.1 gene 13
2.1.3.2 Parental imprinting 14
2.1.3.3 protein 14
2.1.3.4 Regulation of IGF-II expression 15
2.1.3.4.1 RNA 15
2.1.3.4.2 IGF-II levels in the circulation 16
2.1.3.5 Biological effects of IGF-II 16
2.1.4 The IGF receptors 18
2.1.4.1 IGF-I receptor (IGF-IR) 18
2.1.4.2 IGF-II receptor (IGF-IIR) 19
2.1.5 The IGF-binding proteins (IGFBPs) and the acid-labile subunit (ALS) 20
2.1.6 The IGF-binding protein-related proteins 21
2.2 Genetically engineered mouse models for IGF-I and IGF-II 21
2.2.1 Overview 21
2.2.2 IGF-I deficient mice 23
2.2.3 IGF-II transgenic mi 24

3 Animals, Materials and Methods 26
3.1 Animals 26
3.1.1 IGF-I knockout mice 26
3.1.2 PEPCK-IGF-II transgenic mice 26
3.1.3 Crossbreeding 27
3.1.4 Animal husbandry 29
3.2 Mouse genotyping 29
3.2.1 Proteinase K digests of mouse tail tips 29
3.2.2 Determination of the DNA concentration 30
3.2.3 Principle of the Polymerase Chain Reaction (PCR) 30 II Contents
__________________________________________________________________________________________
3.2.4 PCR protocol for detecting the IGF-I knockout sequence 31
3.2.5 PCR protocol for detecting the PEPCK-IGF-II transgene 33
3.3 Evaluation of gene expression at RNA levels 35
3.3.1 RNA extraction 35
3.3.2 Reverse Transcription PCR (RT-PCR) 36
3.3.3 β-Actin PCR 37
3.3.4 PEPCK-IGF-II PCR 38
3.4 Evaluation of gene expression at the protein level 38
3.4.1 Blood serum collection 38
3.4.2 Western ligand and immunoblot analysis 39
3.4.2.1 SDS-PAGE 39
3.4.2.2 Electroblotting 40
3.4.2.3 Westernl ligand blot 41
3.4.2.4 immuno 42
3.4.3 Radioimmunoassay (RIA) 43
3.5 Analysis of body and organ growth
3.6 Bone parameters 44
3.6.1 Bone preparation 44
3.6.2 histology 45
3.6.3 mineral density measurements 45
3.7 Statistical analysis 45
3.8 Reagents and materials 46

4 Results 49
4.1 Generation of double mutant mice 49
4.2 Expression analysis 50
4.2.1 Measurement of circulating levels of IGF-I and IGF-II 50
4.2.2 RT-PCR 51
4.3 Body weight gain 52
4.4 Organ analysis 55
4.5 Analysis of geometric and structural bone parameters 58
4.6 Western ligand blot of IGFBPs 61
4.7 Western immunoblot for growth hormone (GH) 62

5 Discussion 64
5.1 Generation of double mutant mice 64
5.2 Expression analysis 65
5.2.1 Circulating levels of IGF-I and IGF-II 65
5.2.2 Onset of PEPCK-IGF-II expression 66
5.3 Body weight gain 66
5.4 Organ analysis 67
5.5 Bone parameters 68
5.6 Western ligand blot of IGFBPs 70
5.7 Growth hormone Western immunoblot 70
5.8 Final considerations 71

6 Summary 73

7 Zusammenfassung 75

8 Bibliography 7 III
Abbreviations

A Amper
ACTH adrenocorticotropic hormone
ALS acid-labile subunit
APS ammonium persulfate
AU arbitrary unit
BKVI bovine keratin 10 gene
BMD bone mineral density
bp base pair
cDNA complementary DNA
cm centimeters
cpm counts per minute
DEPC diethylpyrocarbotate
DNA desoxyribonucleic acid
dNTP bonucleoside triphosphates
DTT dithiothriteol
e embryonic stage
E exon
EDTA ethylene diamine tetraacetic acid
ES bryonic stem
g gram relative centrifugal force (RCF)
GH growth hormone
h human
IGF-I; -II insulin-like growth factor-I; -II
IGFBP IGF binding protein
IGFBP-rP IGFBP related
IGF-I, -IIR IGF-I, -II receptor
kb kilobase
kDa kilodalton
l liter
LID liver-specific igf1 gene-deletion
M molar, marker
m urine
M-6-P mannose-6-phosphate
mA illiampere
mg milligram
mlilliliter
MLV urine leucemia virus
mm illimeter
mRNA messenger RNA
MSA multiplication stimulating activity
MUP major urinary protein IV
NSILA nonsuppressible insulin-like activity
ng nanogram
nn neonatal
NRL nose-rump-length
OD optical density
P promotor
PBS phosphate-buffered saline
p.c. post coitum
PCR polymerase chain reaction
PDGF platelet-derived growth factor
PEPCK phosphoenolpyruvate carboxykinase
pn postnatal
pQCT peripheral quantitative computed tomography
RIA radioimmunoassay
RNA ribonucleic acid
rpm revolution per minute
RT-PCR reverse transcription PCR
SDS sodium dodecyl sulfate
TAE Tris-acetate-EDTA
TBS Tris-buffered saline
TEMED tetramethylethylendiamine
tg transgenic
TRAMP transgenic adenocarcinoma of mouse
U Unit
UV ultraviolet
V volt
wt wildtype
µg micrograms
µl microliters
1 Introduction and objectives
__________________________________________________________________________________________
1. Introduction and objectives

The size of an animal depends on the number and volume of the cells it contains, with some
contribution by extracellular matrix and fluids. In mammals, growth (increase in size) begins
at pre-implantational embryonic stages and lasts until a steady state is reached postnatally.
Appropriate growth is controlled by hormones and growth factors regulating cellular signaling
pathways.

In mice, the insulin-like growth factor system has been unequivocally identified as the major
determinant of both embryonic and postnatal (here modulated by the growth hormone - GH)
growth (Lupu et al., 2001). The IGF system consists of two ligands (IGF-I and IGF-II), two
receptors (IGF-IR and IGF-IIR) and six high affinity IGF binding proteins (IGFBP-1 to -6).
The single chain peptide growth factors IGF-I and -II are produced by several tissues and
function in an autocrine/paracrine fashion and, since they circulate in the plasma bound to the
IGFBPs, as classical hormones. Despite their structural homology, each growth factor has