Generation of transgenic mice with regulated expression of manganese superoxide dismutase (MnSOD) [Elektronische Ressource] / by Tomasz Loch

V. A. Motzart

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 41
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Generation of transgenic mice with regulated expression
of manganese superoxide dismutase (MnSOD)


Doctoral Thesis




submitted to
Mathematisch-Naturwissenschaftlich-Technische Fakultät
Martin-Luther-Universität Halle-Wittenberg



by
Tomasz Loch
born 22.11.1971 in Siemianowice Śl ąskie, Poland


Reviewers:
1. Prof. Thomas Braun
2. Prof. Thomas Noll

Datum der Verteidigung: 15.09.2006, Halle (Saale)

urn:nbn:de:gbv:3-000010674
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000010674]Contents
1 INTRODUCTION..................................................................................................1
1.1 Free radicals, oxidative stress and antioxidant enzymes ................................................ 1
1.1.1 Free radicals............................................................................................................. 1
1.1.2 Free radicals in physiology...................................................................................... 2
1.1.3 Oxidative stress........................................................................................................ 3
1.1.4 Antioxidant enzymes (AOEs).................................................................................. 3
1.1.4.1 Copper zinc superoxide dismutase (CuZnSOD) .............................................. 4
1.1.4.2 Extracellular superoxide dismutase (ECSOD) ................................................. 5
1.1.4.3 Manganese superoxide dismutase (MnSOD) ................................................... 5
1.1.4.4 Catalase (CAT) ................................................................................................. 6
1.1.4.5 Glutathione peroxidase (GPx) .......................................................................... 6
1.1.4.6 Other antioxidants............................................................................................. 7
1.2 Manganese superoxide dismutase (MnSOD) ................................................................. 8
1.2.1 Gene structure and regulation of transcription ........................................................ 8
1.2.2 Post-transcriptional regulation of MnSOD activity................................................. 9
1.2.3 Effects of MnSOD deficiency ............................................................................... 10
1.2.4 Effects of MnSOD overexpression........................................................................ 12
1.2.5 MnSOD in cancer .................................................................................................. 14
1.2.6 Aims of the project ................................................................................................ 16
2 MATERIAL AND METHODS.............................................................................17
2.1 Material......................................................................................................................... 17
2.1.1 Reagents................................................................................................................. 17
2.1.2 Specific reagents.................................................................................................... 17
2.1.3 Kits 19
2.1.4 Oligonucleotides 19
2.1.5 Antibodies.............................................................................................................. 21
2.1.6 Bacterial strains ..................................................................................................... 21
2.1.7 Plasmids and vectors ............................................................................................. 22
2.1.8 Cell lines................................................................................................................ 23
2.1.9 Mice strains............................................................................................................ 23
2.2 Methods ........................................................................................................................ 24
2.2.1 Generation of cell culture and targeting constructs ............................................... 24
2.2.1.1 Genomic library screening.............................................................................. 24
IContents
2.2.1.2 Mapping of the MnSOD locus........................................................................ 24
2.2.1.3 Generation of cell culture construct pTRE2hyg/SOD2ex .............................. 25
2.2.1.4 Generation of targeting construct pTG/TRE .................................................. 25
2.2.1.5 G/TRE tetR........................................... 26
2.2.1.6 Principles of Tetracycline-regulated expression system................................. 27
2.2.2 Cell culture methods.............................................................................................. 28
2.2.2.1 Basic maintenance .......................................................................................... 28
2.2.2.2 Primary fibroblast culture............................................................................... 29
2.2.2.3 Transient transfections.................................................................................... 29
2.2.2.3.1 Calcium phosphate ................................................................................. 29
2.2.2.3.2 Electroporation ....................................................................................... 30
2.2.2.4 Double-stable cell line generation .................................................................. 30
2.2.2.5 Mouse embryonic stem cells culture .............................................................. 31
2.2.2.5.1 Preparation of mitomycin C treated MEFs............................................. 31
2.2.2.5.2 Basic maintenance .................................................................................. 32
2.2.2.5.3 Electroporation of ES cells..................................................................... 32
2.2.2.5.4 Isolation of recombinant ES cell clones ................................................. 33
2.2.2.5.5 DNA extraction from ES cells................................................................ 33
2.2.3 ROS measurement by Fluorescence Activated Cell Sorting (FACS) ................... 34
2.2.4 AOE’s activity methods......................................................................................... 34
2.2.4.1 Protein extraction............................................................................................ 34
2.2.4.2 Superoxide Dismutase (SOD) ........................................................................ 35
2.2.4.2.1 SOD activity gel ..................................................................................... 35
2.2.4.2.2 Cytochrome C method............................................................................ 35
2.2.4.3 Catalase (CAT) ............................................................................................... 36
2.2.4.4 Glutathione Peroxidase (GPX) ....................................................................... 36
2.2.5 General PCR and RT-PCR methods...................................................................... 37
2.2.5.1 Reverse transcription (RT) ............................................................................. 37
2.2.5.2 Polymerase chain reaction (PCR)................................................................... 38
2.2.5.3 Reverse transcription - Polymerase chain reaction (RT-PCR)....................... 38
2.2.6 Genotyping of recombinant ES cells and transgenic mice .................................... 39
2.2.6.1 Tail DNA isolation ......................................................................................... 39
2.2.6.2 PCR................................................................................................................. 39
2.2.6.3 Southern blot hybridization ............................................................................ 42
IIContents
2.2.7 Tissue sections....................................................................................................... 43
2.2.7.1 Paraffin embedding and sectioning ................................................................ 43
2.2.7.2 Cryosectioning................................................................................................ 43
2.2.8 In situ hybridization............................................................................................... 43
2.2.8.1 Probe synthesis 43
2.2.8.2 ........................................................................................ 44
2.2.9 Senescence Associated β-Galactosidase staining (SA- β-Gal)............................... 45
2.2.10 Immunoprecipitation ............................................................................................. 45
2.2.11 In-situ anti-myc staining 45
2.2.12 Western blotting .................................................................................................... 46
2.2.13 Northern blotting ................................................................................................... 47
2.2.14 Echocardiography.................................................................................................. 47
2.2.15 Statistics...............................