Generation and analysis of a Pink1 deficient mouse model for Parkinson's disease [Elektronische Ressource] / Anne Henriette Janice Röthig

Anne Manning

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Entwicklungsgenetik




Generation and analysis of a Pink1 deficient mouse
model for Parkinson’s disease


Anne Henriette Janice Röthig




Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum
Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen
Universität München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. K. Schneitz
Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst
2. Univ.-Prof. Dr. A. Gierl

Die Dissertation wurde am 08.03.2010 bei der Technischen Universität
München eingereicht und durch die Fakultät Wissenschaftszentrum
Weihenstephan für Ernährung, Landnutzung und Umwelt am 29.07.2010
angenommen.















Für meine Eltern









TABLE OF CONTENTS

1 ABSTRACT................................................................................... 1
2 INTRODUCTION............................................. 5
2.1 MORBUS PARKINSON ................................................................................5
2.1.1 Molecular mechanisms implied in Parkinson’s disease ...................6
2.1.1.1 Protein degradation and the ubiquitin proteasome system
(UPS)...............................................................................6
2.1.1.2 Mitochondrial impairment and oxidative stress .........................7
2.1.1.3 Microtubules and cytoskeletal transport ................................. 10
2.1.2 Familial Parkinson’s disease ......................................................... 11
2.2 PTEN INDUCED KINASE 1 (PINK1) .......................................................... 19
2.2.1 Pink1 and apoptosis ...................................................................... 22
2.2.2 Pink1 deficient Drosophila models ................................................ 22
2.2.3 Pink1 mouse models ..................................................................... 23
2.2.4 Pink1 and mitochondrial dynamics................................................ 24
2.3 AIM OF STUDY........................................................................................ 26
3 MATERIAL.................................................................................. 27
3.1 INSTRUMENTS........................................................................................ 27
3.2 CHEMICALS............................................................................................ 29
3.3 COMMON AND STOCK SOLUTIONS............................................................ 32
3.4 CONSUMABLES AND OTHERS................................................................... 33
3.5 MOLECULAR BIOLOGY............................................................................. 34
3.5.1 Kits for molecular biology .............................................................. 34
3.5.2 Work with bacteria ......................................................................... 34
3.5.2.1 E.coli bacteria strain ............................................................... 34
3.5.2.2 Solutions ................................................................................. 34
3.5.3 Solutions for Southern blot analysis.............................................. 35
3.5.4 Western blot analysis .................................................................... 35
3.5.4.1 Solutions ................................................................................. 35
3.5.4.2 Antibodies ............................................................................... 36
3.5.5 HPLC analysis ............................................................................... 37
3.5.6 Mitochondria extraction ................................................................. 37
3.5.7 Enzymes........................................................................................ 37
3.5.8 Vectors and plasmids .................................................................... 38
3.5.9 Oligonucleotides ............................................................................ 39
I
3.5.9.1 Oligonucleotides for PCR amplification...................................39
3.6 HISTOLOGICAL METHODS.........................................................................40
3.6.1 Solutions for RNA in situ hybridization on paraffin sections ..........40
3.6.2 Antibodies for Immunohistochemistry............................................40
3.6.3 ies for Immunocytochemistry.............................................41
3.6.4 LacZ staining solutions...................................................................41
3.6.5 Counterstaining solutions...............................................................41
3.7 CELL CULTURE .......................................................................................42
3.7.1 Embryonic stem cell lines...............................................................42
3.7.2 Mouse embryonic fibroblast cell lines ............................................42
3.7.3 Solutions.........................................................................................42
3.8 MOUSE LINES .........................................................................................43
3.8.1 Wild type mice................................................................................43
3.8.2 Cre recombinase expressing mice.................................................43
3.8.3 Transgenic mice.............................................................................43
4 METHODS ..................................................................................44
4.1 MOLECULAR BIOLOGY .............................................................................44
4.1.1 Cloning and work with plasmid DNA..............................................44
4.1.1.1 Preparation of plasmid DNA....................................................44
4.1.1.2 Restriction digestion of plasmid DNA......................................44
4.1.1.3 Separation and isolation of DNA fragments............................45
4.1.2 Analysis of genomic DNA...............................................................45
4.1.2.1 Isolation of genomic DNA........................................................45
4.1.2.2 Southern Blot analysis.............................................................45
4.1.2.3 Polymerase Chain Reaction....................................................47
4.1.3 Analysis of RNA .............................................................................48
4.1.3.1 Sample preparation and isolation of RNA...............................48
4.1.3.2 Reverse transcription of mRNA into cDNA .............................49
4.1.4 Analysis of protein..........................................................................49
4.1.4.1 Western blot analysis ..............................................................49
4.1.4.2 Sample preparation for HPLC analysis...................................50
4.1.4.3 Quantification of neurotransmitters via HPLC analysis...........51
4.2 EMBRYONIC STEM (ES) CELL CULTURE ....................................................51
4.2.1 Preparation of feeder cells .............................................................52
4.2.2 Splitting of ES cells ........................................................................52
4.2.3 Freezing and thawing of ES cells...................................................53
4.2.4 Electroporation of ES cells.............................................................54
4.2.5 Selection and picking of recombined clones..................................55
4.2.6 Screening for positive homologous recombination........................56
4.3 MOUSE EMBRYONIC FIBROBLAST (MEF) CELL CULTURE............................56
4.3.1 Generation of MEF cell lines..........................................................57
II
4.3.2 Splitting of MEF cells..................................................................... 58
4.3.3 Freezing and thawing of MEF cells ............................................... 58
4.3.4 Immortalization of MEF cell lines................................................... 58
4.3.4.1 3T3 cells.................................................................................. 58
4.3.4.2 SV40 largeT antigen transfection ........................................... 59
4.3.5 Isolation of mitochondria from MEF cells ...................................... 60
4.4 LENTIVIRUSES........................................................................................ 60
4.4.1 Generation of lentiviruses.............................................................. 60
4.4.2 Viral infection of MEF cell lines ..................................................... 61
4.4.3 Determination of virus titer............................................................. 62
............................................................................... 62 4.5 ANIMAL HUSBANDRY
4.5.1 Animal facilities.............