Functional analysis of insulin-like growth factor binding protein-4 and -6 in transgenic mice [Elektronische Ressource] / by Rui Zhou

Zhou Huashan, Sandrine Rui

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Institute of Molecular Animal Breeding and Biotechnology, Gene Center
Faculty of Veterinary Medicine, University of Munich
Prof. Dr. Eckhard Wolf
Functional Analysis of Insulin-like Growth
Factor Binding Protein -4 and -6
in Transgenic Mice
Thesis for the attainment of the title of Doctor in Veterinary Medicine
from the Faculty of Veterinary Medicine, University of Munich
by
Rui Zhou
from Hubei, P. R. China
Munich, 2003Aus dem Institut für Tierzucht der Tierärztlichen Fakultät der Universität München
Lehrstuhl für Molekulare Tierzucht und Biotechnologie, Genzentrum
Univ.-Prof. Dr. Eckhard Wolf
Funktionelle Analyse der Insulin-like Growth
Factor-Bindungsproteine -4 und -6
in transgenen Mäusen
Inaugural-Dissertation
zur Erlangung der Tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München
von
Rui Zhou
aus Hubei, VR China
München, 2003Gedruckt mit Genehmigung der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München
Dekan: Univ.-Prof. Dr. R. Stolla
1. Referent: Univ.-Prof. Dr. E.Wolf
2. Referent: Univ.-Prof. Dr. Dr. F. Sinowatz
1. Korreferent: Univ.-Prof. Dr. J. Meyer
2. Korreferent: Univ. Prof. Dr. K. Pfister
3. Korreferentin: Priv.-Doz. Dr. M. Rinder
Tag der Promotion: 18. Juli 2003
Gedruckt mit Unterstützung des Deutschen Akademischen
AustauschdienstesMY FAMILYI Contents
CONTENTS
CONTENTS....................................................................................................................................I
ABBREVIATIONS .......................................................................................................................V
1 INTRODUCTION....................................................................................................... 1
2 REVIEW OF THE LITERATURE........................................................................... 3
2.1 Transgenic technologies in mice for studying gene function........................ 3
2.1.1 Conventional transgenic technologies in mice............................................................ 4
2.1.1.1 Pronuclear DNA microinjection.................................................................................... 4
2.1.1.2 Targeted mutagenesis in mice ....................................................................................... 5
2.1.1.2.1 Knockout........................................................................................................................ 5
2.1.1.2.2 Knockin.......................................................................................................................... 6
2.1.2 Conditional transgenic technologies in mice .............................................................. 6
2.1.2.1ional overexpression........................................................................................... 7
2.1.2.2 Conditional knockout .................................................................................................... 7
2.1.3 Gene-trap mutagenesis in mice 9
2.2 Insulin-like growth factor (IGF) system......................................................... 9
2.2.1 IGF peptides ................................................................................................................. 9
2.2.2 IGF receptors..............................................................................................................11
2.2.3 IGF-binding proteins ................................................................................................. 13
2.3 IGFBP-4 .......................................................................................................... 16
2.3.1 Genomic organization of the IGFBP-4 gene ............................................................ 16
2.3.2 The structure-function relationship of IGFBP-4...................................................... 17
2.3.2.1 IGF-binding.... 18
2.3.2.2 Cell surface association and tissue distribution........................................................... 19
2.3.2.3 Glycosylation............................................................................................................... 20
2.3.2.4 Proteolysis ................................................................................................................... 20
2.3.3 IGFBP-4 expression in vivo and its regulation......................................................... 21
2.3.4ression in vitro and its regulation........................................................ 22
2.3.5 Actions of IGFBP-4.................................................................................................... 25
2.3.5.1 IGF-dependent actions ................................................................................................ 25
2.3.5.2 IGF-independent actions ............................................................................................. 26
2.3.6 Biological significance of IGFBP-4 .......................................................................... 26
2.3.6.1 Reproductive physiology 26
2.3.6.2 Bone formation............................................................................................................ 28
2.3.6.3 Renal pathophysiology 29
2.3.6.4 IGFBP-4 and cancer .................................................................................................... 30
2.4 IGFBP-6 30
2.4.1 Genomic organization of the IGFBP-6 gene ............................................................ 30II Contents
2.4.2 The structure-function relationship of IGFBP-6...................................................... 31
2.4.3 IGFBP-6 expression in vivo and its regulation......................................................... 34
2.4.4ression in vitro and its regulation........................................................ 36
2.4.5 Actions of IGFBP-6.................................................................................................... 37
2.4.6 Biological significance of IGFBP-6 .......................................................................... 38
3 ANIMALS, MATERIALS AND METHODS 40
3.1 Animals............................................................................................................ 40
3.2 Cells and cell culture techniques................................................................... 40
3.2.1 Cells............................................................................................................................. 40
3.2.2 Freezing cell lines....................................................................................................... 41
3.2.3 Thawing cell lines 41
3.3 Construction of expression vectors............................................................... 41
3.3.1 Restriction enzyme digest ........................................................................................... 41
3.3.2 Filling 5’- and 3’-protruding ends............................................................................. 42
3.3.3 Dephosphorylation of 5’-ends.................................................................................... 42
3.3.4 Extraction of DNA fragments from agarose gel ....................................................... 42
3.3.5 Ligation of DNA fragments ....................................................................................... 43
3.3.6 Preparation of competent bacteria 43
3.3.7 Transformation of bacteria........................................................................................ 45
3.3.8 Preparation of minipreps and midipreps................................................................... 46
3.4 Expression vectors.......................................................................................... 46
b3.4.1 The H-2K -mcIGFBP-4 and -6 constructs................................................................ 46
3.4.2 The CMV-mgIGFBP-6 construct .............................................................................. 47
3.5 Transfection of cells ....................................................................................... 48
3.6 Production of transgenic mice....................................................................... 49
3.6.1 Generation of transgenic mice by DNA-microinjection ........................................... 49
b3.6.2ion of H-2K -mcIGFBP-4 SPF transgenic mice by embryo transfer ......... 49
3.7 Identification of transgenic mice................................................................... 49
3.7.1 Polymerase Chain Reaction (PCR) ........................................................................... 49
3.7.1.1 Proteinase K digest of mouse tails............................................................................... 50
3.7.1.2 PCR conditions............................................................................................................ 50
3.7.2 Southern blot ..............................................................................................................51
3.7.2.1 Extraction of DNA from tail biopsies.......................................................................... 51
3.7.2.2 Digestion and transfer of the DNA.............................................................................. 52
3.7.2.3 Radioactive probe labeling.......................................................................................... 53
3.7.2.4 Hybridization, washing and signal