Analysis of cathepsin H-deficient mice and identification of a dense cluster of placenta specific peptidase genes [Elektronische Ressource] / vorgelegt von Taneh Martin Kouadio

Wolfgang

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Introduction
Analysis of cathepsin H-deficient mice
and identification of a dense cluster
of placenta specific peptidase genes









Inaugural-Dissertation
Zur
Erlangung der Doktorwürde
Der Fakultät für Biologie
der Albert-Ludwigs-Universität
Freiburg im Breisgau

















Vorgelegt von
Tanoh Martin Kouadio
aus Grand-Bassam
(Côte d’Ivoire)
2004
IIntroduction
Dekan der Fakultät für Biologie : Prof. Dr. G. Fuchs
Leiter der Arbeit und Referent: Prof. Dr. A. E. Sippel
Betreuer der Arbeit: Prof. Dr. C. Peters


Tag der Verkündung des Prüfungsergebnisses: 03. November 2004
IIntroduction
N’écouter que son coeur
Et vivre jusqu´au bout
Sa légende personnelle
Quelqu´en soit le prix,
Quelqu´en soit le temps
Que cela pourrait coûter.

Paulo Coelho










































II Introduction
I would like to give special thanks Prof. Dr. Christoph Peters for accepting me in her
laboratory and allowing me to do this work.

My gratitude goes to Prof. Dr. A. E. Sippel for supervising of my work and for helping during
the difficult moments.


Specials thanks to Dr. Jan Deussing and Dr. Thomas Reineckel with whose exceptional daily
supervision I was able to complete this work


Many thanks also go to all the personnel of the Peter´s Lab for their kind collaboration during
my stay.

Mes sincères remerciements Miss Brigitte Kost pour sa très éfficace intervention.
























IIIIntroduction














A ma mère chérie,
Ta patience n´aura pas été
Et ne sera jamais vaine









A Nina et Ama,
Désormais j´aurais
Plus de temps.

IVIntroduction
TABLE OF CONTENTS
1 INTRODUCTION............................................................................................................ 1
1.1 Proteolysis.................................................................................................................. 1
1.2 The Lysosomal and Endosomal Compartment .......................................................... 1
1.3 Lysosomal Proteases..................................................................................................2
1.4 Lysosomal Cysteine Proteases...................................................................................3
1.5 Physiological Roles of Lysosomal Cysteine Proteases.............................................. 4
1.6 Pathological Roles......................................................................................................4
1.7 Placenta-specific Cysteine Proteases: "Cathepsin-J-like Enzymes" .......................... 5
1.8 Gene Targeting...........................................................................................................5
1.8.1 Homologous Recombination and Generation of knock-out Mice ..................... 6
2 research statement 9
3 Materials and methods .................................................................................................. 10
3.1 Materials................................................................................................................... 10
3.1.1 Materials for Molecular and Cell Biology ....................................................... 10
3.1.2 Blotting Material..............................................................................................10
3.1.3 Enzymes, Standards and Antibiotics................................................................
3.1.4 Kits for Nucleic Acid ....................................................................................... 11
3.1.5 Radioactive Substance......................................................................................11
3.1.6 Antibody...........................................................................................................11
3.1.7 Substrat.............................................................................................................
3.1.8 Medium............................................................................................................11
3.1.9 Stamm Solution................................................................................................12
3.1.10 Medium for Bacteria........................................................................................
3.1.11 Vector...............................................................................................................12
3.1.12 Bacteria
3.1.13 ES-Cells12
3.1.14 Mouse Strains...................................................................................................13
3.1.15 Material for mouse methods.............................................................................
3.1.16 Material for Histology...................................................................................... 13
3.1.17 Material for LacZ Staining............................................................................... 13
3.1.18 Pac Isolation and Contig Construction............................................................. 13
3.2 Methods.................................................................................................................... 14
3.2.1 Methods of molecular biology ......................................................................... 14
3.2.2 Hybridization....................................................................................................22
3.3 Biochemical Methods...............................................................................................24
3.3.1 Preparation of Lysosomal Extracts ..................................................................
3.3.2 Determination of Protein Concentration “Bio-Rad DC Protein Assay” .......... 25
3.3.3 Cathepsin H Activity........................................................................................ 26
3.3.4 SDS-polyacrylamide Gel Glectrophoresis (SDS-PAGE) of Proteins.............. 26
3.3.5 Transfer of Protein on to Nitrocellulose and Immunodetection (Western Blot)
27
3.4 Embryonic stem (ES) cell culture ............................................................................ 27
3.4.1 Selection of ES-cell clones............................................................................... 27
3.4.2 Trypsinization and Freezing of HM-1 ES Cells............................................... 28
3.5 Mouse Methods........................................................................................................28
3.5.1 Superovulation induced by Hormonal Manipulation .......................................
3.5.2 Mouse Mating..................................................................................................29
3.5.3 Plug Check.......................................................................................................
3.6 Histology.................................................................................................................. 29
VIntroduction
3.6.1 Dehydratation of Histological Samples............................................................ 29
3.6.2 Embedding of Tissues in Paraffin .................................................................... 30
3.6.3 Tissue Sections.................................................................................................30
3.6.4 Tissue Staining
3.7 LacZ-staining...........................................................................................................31
3.8 Screening of Mice ....................................................................................................
3.9 Statistical Analysis...................................................................................................33
4 Results ............................................................................................................................. 34
4.1 Gene Targeting and Generation of Cathepsin H-Deficient Mice............................. 34
4.2 Functional Inactivation of the Cathepsin H gene ..................................................... 35
4.2.1 Northern-blot Analysis.....................................................................................35
4.2.2 Western-Blot 36
4.2.3 Cathepsin H Enzyme Activity Assay............................................................... 37
4.3 Phenotype of Cathepsin H-Deficient Mice. ............................................................. 38
4.4 Localization of mCathepsin H Expression 39
4.4.1 mCTSH Expression in Lung .................................................................