Up-regulation of sterol regulatory element binding protein 2 (Srebp2)-mediated cholesterol biosynthesis in prion infected cells [Elektronische Ressource] / Christian Erich Bach

Bach

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 13
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Tierhygiene


Up-regulation of sterol regulatory element binding
protein 2 (Srebp2)-mediated cholesterol biosynthesis in
prion infected cells

Christian Erich Bach

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. D. Langosch
Prüfer der Dissertation:
1. Univ.-Prof. Dr. Dr. h. c. J. Bauer
2. Priv.-Doz. Dr. J. Beckers
3. Univ.-Prof. Dr. H. Schätzl



Die Dissertation wurde am 08.06.2009 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 01.11.2009 angenommen.




























Dedicated to my parents
for all their help and support







Table of contents
Summary .................................................................................................................................... 1 
English version ................................................................................................................... 1 
Deutsche Version ............................................................................................................... 3 
1.  Introduction ........................................................................................................................ 5 
1.1  Prion diseases in animals ............................................................................................ 7 
1.2  Human prion diseases ................................................................................................. 9 
C1.3  The cellular prion protein PrP ................................................................................ 13 
C Sc1.4  Structural and biochemical features of PrP and PrP ............................................ 17 
C Sc1.5  Models for conversion of PrP to PrP ................................................................... 19 
1.6  Prion strains and the species barrier ......................................................................... 21 
1.7  Prion cell culture models .......................................................................................... 22 
1.8  Cellular response (in vivo and in vitro) upon prion infection .................................. 23 
Aim of this PhD thesis ............................................................................................................. 25 
2.  Material and Methods ....................................................................................................... 26 
2.1  Materials ................................................................................................................... 26 
2.1.1  Chemicals ............................................................................................................. 26 
2.1.2  Kits ....................................................................................................................... 27 
2.1.3  Cell lines ............................................................................................................... 28 
2.1.4  Cell culture media and additives .......................................................................... 28 
2.1.5  Oligodeoxynucloetides ......................................................................................... 29 
2.1.6  Enzymes and antibodies ....................................................................................... 29 
2.1.6.1  Enzymes ....................................................................................................... 29 
2.1.6.2  Antibodies .................................................................................................... 30 
2.1.7  Plasmids and bacterial strains .............................................................................. 31 
2.1.8  Instruments ........................................................................................................... 32 
2.2  Methods .................................................................................................................... 33 
2.2.1  Biological safety ................................................................................................... 33 
2.2.2  Photodensitometric analysis and statistics ........................................................... 34 
2.2.3  Molecular biological methods 34 
2.2.3.1  Transformation of E. coli with plasmid DNA .............................................. 34 
2.2.3.2  Isolation of plasmid DNA in a preparative scale (Maxi-Prep) ..................... 34 
2.2.3.3  Isolation of total cellular RNA ..................................................................... 35 
2.2.3.4  Quantification of nucleic acids 35 
I
2.2.3.5  Polymerase chain reaction (PCR) for amplification of DNA fragments ..... 36 
2.2.3.6  Electrophoretic separation of DNA/RNA (agarose gel electrophoresis) ..... 36 
Separation of DNA ....................................................................................... 37 
Separation of RNA 37 
2.2.3.7  Mouse genome-wide cDNA microarray ...................................................... 37 
DNA microarray (chip) design ..................................................................... 37 
Reverse transcription and fluorescent labelling ........................................... 38 
Data analysis and analysis of differentially expressed genes ....................... 38 
2.2.3.8  Quantitative real-time reverse transcription polymerase chain reaction (Real-
Time PCR) .................................................................................................................... 39 
2.2.3.9  Transient transfection of mammalian cells .................................................. 42 
Transfection of plasmid DNA for luciferase assay ...................................... 42 
Transfection with siRNA ............................................................................. 43 
2.2.3.10  Luciferase assay ........................................................................................... 43 
2.2.3.11  Transient knock-down of Srebp2 and Ldlr expression by siRNA ............... 45 
2.2.4  Protein biochemical methods ............................................................................... 46 
C Sc2.2.4.1  Preparation of postnuclear lysates for PrP /PrP detection ........................ 46 
2.2.4.2  Preparation of pre-nuclear lysates for Serbp2 detection .............................. 46 
2.2.4.3  Proteinase K (PK) digestion of post-nuclear lysates .................................... 47 
2.2.4.4  Bradford assay for determination of protein concentration ......................... 47 
2.2.4.5  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 48 
2.2.4.6  Western blot (Immunoblot) .......................................................................... 49 
2.2.4.7  Thin layer chromatography (TLC) for determination of cellular cholesterol
level ...................................................................................................................... 50 
2.2.4.8  Protein detection with immunofluorescence microscopy ............................ 51 
2.2.4.9  Fluorescent-activated cell sorting (FACS) analysis ..................................... 52 
2.2.5  Culturing and passaging of eukaryotic cell lines ................................................. 52 
2.2.5.1  Cultivation and passaging of mammalian cell lines 52 
2.2.5.2  Subcloning of N2a cells ............................................................................... 53 
2.2.5.3  Preparation of primary neurons and astrocytes ............................................ 53 
2.2.5.4  Cryoconservation of cells ............................................................................. 54 
2.2.5.5  Determination of cell number ...................................................................... 54 
2.2.5.6  Preparation of prion containing and normal brain homogenates ................. 54 
2.2.5.7  Infection of cells with prions ........................................................................ 55 
II
3.  Results .............................................................................................................................. 56 
3.1  Isolation of a N2a cell clone highly susceptible to prion infection .......................