In vivo modulation of integrin linked kinase using transgenic mice [Elektronische Ressource] / by Chiraz El-Aouni

Faculty of Biology, Ludwig-Maximilians University of Munich






In vivo modulation of integrin linked kinase
using transgenic mice




Thesis for the attainment of the doctoral degree
From the Faculty of Biology, Ludwig-Maximilans University, Munich




By
Chiraz El-Aouni
Tunesia, 2006


Fakultät für Biologie der Ludwig-Maximilian-Universität
München





In vivo modulation of integrin linked kinase
using transgenic mice




Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität
München Zur Erlangung des Dr.rer.nat.




Vorgelegt von
Chiraz El-Aouni
Tunesien, 2006

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Herrn Prof. Dr. Matthias betreut.







Ehrenwörtliche Versicherung
Ich versichere hiermit ehrenwörtlich, dass die Dissertation von mir selbstständig, ohne
unerlaubte Hilfe angefertigt ist.

München, am 20.03.2006


Chiraz El-Aouni



Decan Prof. Dr. Jürgen Soll

First Examiner: Dr. Habil Angelika Böttger
Second Examiner: Prof. Dr. Harry MacWilliams
Co-Examiners: Prof. Dr. Michael Schleicher
Prof. Dr. Dirk Eick
Supervisor: Prof. Dr. Matthias Kretzler


Date of Submission: 20.03.2006
Date of Oral Exam: 22.09.2006






















Dedicated to my mama





Table of contents
Table of contents
TABLE OF CONTENTS............................................................................................................... I
ABBREVIATIONS .......................................................................................................................V
1. INTRODUCTION......................................................................................................................2
1.1. The kidney.............................................. 2
1.1.1. The glomerular filtration barrier...................................................................................................................... 4
1.1.2. Glomerular basement membrane..................................................................................................................... 6
1.1.3. Podocyte.......................................... 7
1.1.3.1. The sole of the podocyte foot process...................................................................................................... 9
1.1.3.2. Podocyte proteins..................................................................................................................................... 9
1.1.3.3. The slit diaphragm ................................................................................................................................. 11
1.2. Glomerular disease and podocyte ...................................................................................................................... 11
1.2.1. IgA nephropathy............................................................................................................................................ 12
1.2.2. Hereditary nephritis- Alport syndrome.......................................................................................................... 12
1.2.3. Diabetic nephropathy..................................................................................................................................... 12
1.2.4. Glomerulosclerosis and Focal Segmental Glomerulosclerosis...................................................................... 13
1.2.5. Minimal change disease ................................................................................................................................ 13
1.2.6. Conginital nephrotic syndrome.......................... 14
1.3. Integrins ............................................................................................................................................................... 14
1.3.1. Signaling machinery of Integrins .................................................................................................................. 15
1.4. The extracellular matrix................................... 15
1.5. The cytoskeleton.................................................................................................................................................. 16
1.6. Integrin linked kinase (ILK) .............................................................................................................................. 17
1.6.1. ILK structure ................................................................................................................................................. 17
1.7. The kinase activity of ILK .................................................................................................................................. 19
1.7.1. ILK expression in human cancers ................................................................................................................. 20
1.7.2. Role of ILK in relevance to nephrophathy .................................................................................................... 20
1.7.3. ILK in kidney disease.................................................................................................................................... 21
1.8. Transgenic mice........................................ 22
1.8.1. Conventional transgenic mice ....................................................................................................................... 22
1.8.1.1. Pronuclear injection............................................................................................................................... 22
i Table of contents
1.8.1.2. Gene targeting........................................................................................................................................ 23
1.8.2. Conditional transgenic mice .......................................................................................................................... 24
1.8.2.1. Conditional knockout............................................................................................................................. 24
2. AIM OF THE STUDY .............................................................................................................26
3. MATERIALS AND METHODS28
3.1. Animals experimentation.................................................................................................................................... 28
3.2. Generation of ILK deficient mice in podocyte.................................................................................................. 28
3.3. Identification of transgenic mice........................................................................................................................ 29
3.3.1. Polymerase chain reaction (PCR)......................... 29
3.3.1.1. Proteinase K digest of mouse tails......................................................................................................... 29
3.3.1.2. PCR conditions ...................................................................................................................................... 29
3.3.2. Agarose gel electrophoresis............................................................................................. 30
3.3.3. Isolation and elution of DNA fragments from agarose gel............................................................................ 31
3.3.4. Quantification of DNA .................................................................................................................................. 32
3.3.5. Enzymatic digestion of DNA ........................................................................................................................ 32
3.3.6. DNA dephosphorylation............................. 32
3.3.7. DNA ligation ................................................................................................................................................. 33
3.3.8. Electroporation of E. coli............................................................................................................................... 33
3.4. DNA extraction and purification ....................................................................................................................... 34
3.4.1. Mini preparation ............................................................................................................................................ 34
3.4.2. Midi preparation 35
3.5. Protein analysis.................................................................................................................................................... 35
3.5.1. Extraction of protein from cells..................................................................................................................... 35
3.5.2. Extraction of proteins from tissues...........................................................................................................