The functional relevance of MKK4 and MKK7 splice variants in neural cells [Elektronische Ressource] / vorgelegt von Wiebke Häusgen

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Sujets

• Biologie

Informations

• Publié par: christian-albrechts-universitat_zu_kiel
• Publié le: 01 janvier 2010
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

The functional relevance of MKK4 and MKK7
splice variants in neural cells









Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel


vorgelegt von

Dipl.-Biol. Wiebke Häusgen

Kiel
2010



























Referent: Prof. Dr. Thomas Herdegen
Korreferent: Prof. Dr. Manuela Dittmar
Tag der mündlichen Prüfung: 30. November 2010
Zum Druck genehmigt: 30. November 2010
gez. Prof. Dr. Lutz Kipp, Dekan

























Meinen ElternTable of contents I
Table of contents
1 Introduction.............................................................................. 1
1.1 Mitogen-activated protein kinase kinase 4 ..................................................................2
1.2 Mitogen-activated protein kinase kinase 7 ..................................................................4
1.3 The c-Jun N-terminal kinases......................................................................................6
1.4 Regulation of JNKs by MKK4 and MKK7.................................................................8
1.5 PC12 cells- a versatile model for peripheral mammalian neurons..............................9
1.6 Aims of the study.......................................................................................................10
2 Materials and methods.......................................................... 12
2.1 Materials ....................................................................................................................12
2.2 Centrifuges, cell culture equipment and sterilization ................................................16
2.3 Culture, staining, stimulation and transfection of cells .............................................17
2.3.1 PC12 cells ................................................................................................................... 17
2.3.2 Electronic cell counting............................................................................................... 19
2.3.3 Trypan blue viability staining ..................................................................................... 19
2.3.4 Flow cytometric analysis............................................................................................. 20
2.3.5 5-bromo-2-deoxyuridine (BrdU) incorporation assay................................................. 20
2.3.6 Differentiation............................................................................................................. 20
2.3.7 Neurite outgrowth ....................................................................................................... 21
2.3.8 Coomassie blue staining.............................................................................................. 21
2.3.9 Incubation with inhibitors and stressors...................................................................... 21
2.3.10 Transfection ................................................................................................................ 22
2.4 Processing of PC12 cells for RNA extraction ...........................................................23
2.4.1 Experimental precautions to minimize RNA degradation .......................................... 23
2.4.2 Harvesting cells........................................................................................................... 23
2.5 Isolation of RNA .......................................................................................................23
2.5.1 RNA extraction ........................................................................................................... 23
2.5.2 RNA quantification and quality control...................................................................... 24
2.5.3 RNA agarose gel ......................................................................................................... 24
2.6 Reverse transcription polymerase chain reaction (RT-PCR) ....................................25
2.6.1 Reverse transcription of mRNA.................................................................................. 25
2.6.2 PCR ............................................................................................................................. 26
2.7 TaqMan Real-Time-quantitative PCR (QRT-PCR) ..................................................28
2.8 Cloning of cDNA fragments......................................................................................29 Table of contents II
2.8.1 General principles and procedure................................................................................ 29
2.8.2 Production of competent E. coli DH5α cells............................................................... 29
2.8.3 Generation of cloning primers and selection of restriction enzymes .......................... 30
2.8.4 Amplification and purification of a PCR product ....................................................... 31
2.8.5 Restriction digestion.................................................................................................... 32
2.8.6 Purification of a digestion reaction ............................................................................. 33
2.8.7 Ligation of DNA fragments into the pEGFP-C3 vector ............................................. 33
2.8.8 Heat-shock transformation of E. coli DH5α cells....................................................... 34
2.8.9 Preparation of plasmid DNA....................................................................................... 34
2.8.9.1 Mini preparation of plasmid DNA................................................................ 34
2.8.9.2 Midi preparation of plasmid DNA................................................................ 35
2.8.9.3 Endotoxin-free maxi preparation of plasmid DNA ...................................... 35
2.8.9.4 Plasmid purification...................................................................................... 36
2.8.10 Sequence verification.................................................................................................. 36
2.9 Denaturing protein extraction and protein quantification..........................................36
2.9.1 Harvesting cells........................................................................................................... 36
2.9.2 Denaturing extraction of whole cell proteins .............................................................. 37
2.9.3 Denaturing extraction of nuclear proteins................................................................... 37
2.9.4 Non-denaturating extraction of whole cell proteins.................................................... 37
2.9.5 Non-denaturating extraction of whole cell proteins for immunoprecipitations .......... 38
2.9.6 Determination of protein concentrations..................................................................... 38
2.9.7 Immunoprecipitation (IP) and immunodepletion (ID) ................................................ 39
2.9.8 Chromatin immunoprecipitation (ChIP) ..................................................................... 39
2.10 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
Western blot...............................................................................................................40
2.10.1 SDS-PAGE.................................................................................................................. 40
2.10.2 Western blot ................................................................................................................ 42
2.10.3 Stripping of Western blot membranes......................................................................... 45
2.10.4 Ponceau S staining of Western blot membranes ......................................................... 45
2.11 Statistical analysis and replication of the experiments..............................................45
3 Results ..................................................................................... 47
3.1 Stable transfection of PC12 cell lines........................................................................47
3.1.1 Cloning of MKK4 and MKK7 expression vectors...................................................... 47
3.1.2 Stable transfection of PC12 cells with MKK4 and MKK7 constructs........................ 52
3.2 Functional analysis of MKK7γ1 in naïve PC12 cells................................................53
3.2.1 MKK7γ1 under normal cell growth conditions........................................................... 53 Table of contents III
3.2.1.1 Characterisation of MKK7γ1-transfected PC12 cells................................... 53
3.2.1.2 Cell proliferation .......................................................................................... 56
3.2.1.3 Apoptotic proteins ........................................................................................ 58
3.2.1.4 mRNA levels of targets ......................................