Mouse models targeted for the tissue inhibitor of metalloproteinases-3 (TIMP3) [Elektronische Ressource] : molecular and functional dissection of Sorsby fundus dystrophy (SFD) / vorgelegt von Marton Fogarasi

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 17
• Langue: Deutsch

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Mouse models targeted for the tissue inhibitor of
metalloproteinases-3 (TIMP3) - molecular and functional
dissection of Sorsby fundus dystrophy (SFD)



Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)
der Naturwissenschaftlichen Fakultät III – Biologie und Vorklinische Medizin der
Universität Regensburg



Vorgelegt von
Marton Fogarasi
aus Rumänien






Regensburg
April, 2009















Promotionsgesuch eingereicht am: 22.04.2009
Tag der mündlichen Prüfung:
Die vorliegende Arbeit wurde von Prof. Dr. Bernhard Weber angeleitet.

Prüfungsausschuss:
Vorsitzender: Prof. Dr. Herbert Tschochner
Erstgutachter: Prof. Dr. Rainer Deutzmann
Zweitgutachter: Prof. Dr. Bernhard Weber
Prüfer aus der Fakultät: Prof. Dr. Ernst Tamm
Vertreter des Drittprüfers: Prof. Dr. Stephan Schneuwly









Eidesstattliche Erklärung
(zu § 6 Abs. 1, Nr. 3 der Promotionsordnung der Naturwissenschaftlichen Fakultäten
der Universität Regensburg)

Ich erkläre hiermit an Eides statt, daß ich die vorligende Arbeit ohne unzulässige Hilfe
Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe;
die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind
unter Angabe des Literaturzitats gekennzeichnet.

Weitere Personen waren an der inhaltlich-materiellen Herstellung der vorliegenden
Arbeit nicht beteiligt. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe eines
Promotionsberaters oder anderer Personen in Anspruch genommen. Niemand hat von
mir weder unmittelbar noch mittelbar geldwerte Leistungen für Arbeiten erhalten, die
im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.

Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form
einer anderen Prüfungsbehörde vorgelegt.


Regensburg, 22.04.2009

Table of contents

1. Introduction.........................................................................................................................1
1.1 Sorsby fundus dystrophy .............................................................................................1
1.2 Structure and function of the retina .............................................................................1
1.3 The extracellular matrix (ECM) ..................................................................................3
1.4 Mouse models of TIMP3 mutations ............................................................................6
1.4.1 TIMP3 knock-out mouse .......................................................................................6
1.4.2 TIMP3 Ser156Cys knock-in mouse.......................................................................7
1.5 Structure of tissue inhibitors of metalloproteinases (TIMPs)......................................7
1.6 Biological functions of TIMP3....................................................................................8
1.6.1 Inhibition of MMPs by TIMP3..............................................................................8
1.6.2 Regulation of the activity of ADAMs by TIMP3................................................10
1.6.3 TIMP3 and ADAMTS inhibition.........................................................................14
1.6.4 Antiangiogenic properties of TIMP3...................................................................17
1.6.5 Proapoptotic activity of TIMP3 ...........................................................................18
1.6.6 TIMP3 as tumour suppressor gene ......................................................................19
1.7 Aims of the present study ..........................................................................................19
2. Materials and Methods.....................................................................................................21
2.1 Molecular biology methods .......................................................................................21
2.1.1 Mammalian cell culture and cryopreservation of cells........................................21
2.1.2 Culture and conservation of E. coli strains..........................................................21
2.1.3 Preparation and transformation of E. coli competent cells..................................22
2.1.3.1 Preparation of bacterial competent cells........................................................22
2.1.3.2 Transformation of competent cells with plasmid DNA.................................22
2.1.4 RNA isolation from primary cell culture.............................................................22
2.1.5 Plasmid DNA isolation from E. coli....................................................................22
2.1.6 Gel electrophoresis and DNA purification ..........................................................23
2.1.6.1 Analytical agarose gel electrophoresis23
2.1.6.2 Preparative agarose gel electrophoresis.........................................................23
2.1.7 In vitro modification of DNA ..............................................................................24
2.1.7.1 Site directed mutagenesis...............................................................................24
2.1.7.2 Dephosphorylation of linearized plasmid DNA ............................................24
2.1.7.3 Filling in DNA sticky end24 2.1.7.4 Polymerase chain reaction (PCR) ..................................................................25
2.1.7.5 First-strand cDNA synthesis..........................................................................25
2.1.7.6 Double stranded DNA cleavage with restriction endonucleases ...................26
2.1.7.7 Ligation of DNA fragments...........................................................................26
2.1.7.8 Double stranded DNA sequencing ................................................................26
2.1.7.9 Phenol/chloroform extraction and ethanol precipitation ...............................27
2.1.8 Production of recombinant proteins in E. coli .....................................................28
2.1.8.1 Protein expression in inclusion bodies...........................................................28
2.1.8.2 Recombinant protein refolding ......................................................................29
2.1.9 Recombinant protein expression in mammalian cells .........................................29
2.1.9.1 Transiently transfection.................................................................................30
2.1.9.2 Stable transfection..........................................................................................30
2.2 Protein biochemistry methods ...................................................................................30
2.2.1 Protein purification..............................................................................................30
2.2.1.1 Immobilized metal affinity chromatography (IMAC) ...................................31
2.2.1.2 Glutathione-S-transferase (GST) chromatography........................................31
2.2.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)......................................31
2.2.3 Zymography.........................................................................................................32
2.2.4 Reverse zymography............................................................................................33
2.2.5 Measurement of protein concentration ................................................................34
2.2.5.1 Estimation of protein concentration at 280 nm absorption............................34
2.2.5.2 Estimation of protein concentration by Lowry assay ....................................34
2.2.6 ECM protein preparation .....................................................................................35
2.2.7 Preparation of cell extracts ..................................................................................35
2.2.8 Concentration of protein solution ........................................................................35
2.2.9 Pull-down assay...................................................................................................36
2.3 Immunochemistry methods........................................................................................36
2.3.1 Immunoprecipitation of proteins.........................................................................36
2.3.2 Enzyme-Linked Immunosorbent Assay (ELISA)................................................36
2.3.2.1 Determination of the apparent dissociation constant by ELISA....................37
2.3.2.2 ELISA applied to cells...................................................................................38
2.3.2.3 Competitive ELISA.......................................................................................39
2.3.3 Western blotting40
2.3.4 Immunocytochemistry.........................................................................................40 2.4 Biophys