Expression and function of transglutaminase 1 in the brain [Elektronische Ressource] / vorgelegt von Lars Dolge

Lars Dolge

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2010
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

„Expression and function of transglutaminase 1 in the brain”




Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen
University zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation





vorgelegt von

Diplom Biologe
Lars Dolge
aus Waiblingen







Berichter: Universitätsprofessor Dr. techn. Werner Baumgartner
Universitätsprofessor Dr. rer. nat. Jürgen Bernhagen


Tag der mündlichen Prüfung: 23.02.2010










Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. ii Content

1. Introduction ...................................................................................................................... 1
1.1 The transglutaminase family ................................................................................................ 3
1.2 Structure and catalytic activity of transglutaminases ........................................................ 5
1.3 Transglutaminases in the central nervous system .............................................................. 8
1.4 Aim of the thesis..................................................................................................................... 9
2. Materials and Methods................................................................................................... 10
2.1 Materials............................................................................................................................... 10
2.1.1 Chemicals and enzymes................................................................................................................ 10
2.1.2 Devices.......................................................................................................................................... 12
2.1.3 Kits................................................................................................................................................ 13
2.1.4 Antibodies..................................................................................................................................... 13
2.1.5 Primer............................................................................................................................................ 14
2.1.6 Animals......................................................................................................................................... 14
2.2 Cell culture ........................................................................................................................... 15
2.2.1 General cell culture solutions........................................................................................................ 15
2.2.2 PC12 cell culture........................................................................................................................... 16
2.2.2.1 Maintenance of PC12 cell culture ......................................................................................... 16
2.2.2.2 Transferring PC12 cells to glass cover slips.......................................................................... 17
2.2.2.3 Differentiating PC12 cells..................................................................................................... 17
2.2.2.4 Transfection of PC12 cells .................................................................................................... 18
2.2.2.5 Whole cell current clamp recordings of PC12 cells .............................................................. 18
2.2.3 Sf9 cell culture .............................................................................................................................. 19
2.2.4 Isolation of chicken telencephalic cells......................................................................................... 20
2.2.5 Isolation of murine cerebellar granule cells .................................................................................. 21
2.3 General methods .................................................................................................................. 24
2.3.1 Discontinuous sodium dodecyl sulphate polyacrylamide gel electrophoresis .............................. 24
2.3.2 Coomassie brilliant blue staining of polyacrylamide gels............................................................. 25
2.2.3 Western Blot ................................................................................................................................. 26
2.3.4 Polymerase chain reaction ............................................................................................................ 28
2.3.5 Agarose gel electrophoresis .......................................................................................................... 28
2.4 Immunofluorescence stainings............................................................................................ 30
2.4.1 Immunocytochemical stainings of cultured cells .......................................................................... 30
2.4.2 Cryostatic brain slices and immunohistochemistry....................................................................... 31
2.5 Analysis of transglutaminase activity................................................................................. 33
2.5.1 Biotinylation of transglutaminase target proteins in cell culture................................................... 33
2.5.2 Purification of biotinylated proteins.............................................................................................. 33
2.6 Generation and analysation of recombinant human transglutaminase 1....................... 34
2.6.1 Cloning of TG1 cDNA into pFastBac1......................................................................................... 34
2.6.2 Generation of the recombinant bacmid ......................................................................................... 34
2.6.3 Isolation of recombinant bacmid................................................................................................... 35
2.6.4 Transfecting Sf9 cells and isolating P1 viral stock ....................................................................... 37
2.6.5 Amplifying the baculoviral stock.................................................................................................. 37
2.6.6 Viral plaque assay......................................................................................................................... 38
2.6.7 Expression of recombinant transglutaminase 1............................................................................. 39
2.6.8 Purification of recombinant transglutaminase 1............................................................................ 39
2.6.9 Cross-linking of actin with recombinant transglutaminase 1........................................................ 40



iii 2.7 Cloning of DNA fragments.................................................................................................. 41
2.7.1 Generation of competent cells....................................................................................................... 41
2.7.2 Transformation of competent cells................................................................................................ 43
2.7.3 Analytical plasmid preparation ..................................................................................................... 44
2.7.4 Ligation of DNA fragments into a vector ..................................................................................... 45
2.8 Conditional transglutaminase 1 knock-out mouse............................................................ 46
2.8.1 Assembly of the transglutaminase 1 knock-out vector ................................................................. 46
2.8.2 Analysis of stem cell clones via Southern blot ............................................................................. 46
3. Results ............................................................................................................................. 50
3.1 Transglutaminase 1 expression in the murine brain ........................................................ 50
3.2 Transglutaminase 1 expression in cerebellar granule cell culture .................................. 56
3.3 Transglutaminase activity in neuronal cell cultures......................................................... 59
3.4 Synaptic activity dependent activation of transglutaminases.......................................... 68
3.5 Transglutaminase 1 knock-out mouse ............................................................................... 73
4. Discussion ....................................................................................................................... 76
4.1 Expression of transglutaminase 1 in the murine brain and cerebellar granule cells..... 76
4.2 Transglutaminase 1 knock-out mouse ............................................................................... 80
4.3 Transglutaminase activity in neuronal cell cultures......................................................... 81
4.4 β-Actin is a target for transglutaminase activity in chicken telencephalic cell cultures 84
4.5 Synaptic activity depe