Analysis of the role of the p47 GTPase IIGP1 in resistance against intracellular pathogens [Elektronische Ressource] / vorgelegt von Iana Angelova Parvanova

Brother Arnold

Sujets

• Biologie

Informations

• Publié par: universitat_zu_koln
• Publié le: 01 janvier 2005
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Analysis of the role of the p47 GTPase IIGP1
in Resistance against Intracellular Pathogens





















Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln




vorgelegt von
Iana Angelova Parvanova
aus Bulgarien



Köln 2005















































Berichterstatter: Prof. Dr. Jonathan C. Howard
Prof. Dr.Jens Brüning

Tag der mündlichen Prüfung: 19.7.2005















































To my family

1 INTRODUCTION 1
1.1 Infection and immunity- the never ending battle......................................................................1
1.2 Interferons, the central players in antimicrobial and antiviral immunity..............................3
1.3 Antimicrobial functions of IFN γ.................................................................................................5
1.4 IFN-induced cell-autonomous immunity ...................................................................................6
1.5 IFN-inducible GTPases as cell-autonomous resistance factors................................................6
1.5.1 Mx proteins............................................................................................................................7
1.5.2 p65-kDa GBPs.......................................................................................................................8
1.5.3 Very large inducible GTPases (VLIGs) ................................................................................8
1.5.4 p47 (IRG) GTPases ...............................................................................................................9
1.5.4.1 IIGP1..........................................................................................................................11
1.6 Aims of this study.......................................................................................................................13
2 MATERIALS AND METHODS 14
2.1 Chemicals and reagents.............................................................................................................14
2.1.1 Oligonucleotides..................................................................................................................14
2.1.2 Enzymes ..............................................................................................................................14
2.1.3 Kits ......................................................................................................................................15
2.1.4 Serological reagents.............................................................................................................15
2.1.4.1 Primary antibodies and antisera .................................................................................15
2.1.4.2 Secondary antibodies and antisera .............................................................................15
2.2 Media ..........................................................................................................................................15
2.2.1 Luria Bertani (LB) medium.................................................................................................15
2.2.2 LB agar medium..................................................................................................................15
2.2.3 EF medium ..........................................................................................................................16
2.2.4 ES medium....16
2.2.5 Freezing medium.................................................................................................................16
2.3 Cells and cell lines......................................................................................................................16
2.3.1 Bacterial strains ...................................................................................................................16
2.3.2 Bacterial pathogens .............................................................................................................16

2.3.3 Protozoan parasites..............................................................................................................17
2.3.4 Mammalian cells and cell lines ...........................................................................................17
2.4 Methods ......................................................................................................................................17
2.4.1 Molecular biology ...............................................................................................................17
2.4.1.1 Preparation of competent Cells..................................................................................17
2.4.1.2 Isolation of Plasmid DNA..........................................................................................17
2.4.1.3 Isolation of Genomic DNA from mouse tissues and cells .........................................17
2.4.1.4 Agarose gel electrophoresis purification of DNA fragments from agarose gels........18
2.4.1.5 Quantification of nucleic acids18
2.4.1.6 Polymerase Chain Reaction .......................................................................................19
2.4.1.6.1 General PCR protocol 19
2.4.1.6.2 Mouse typing PCR for IIGP1 deletion 19
2.4.1.7 Cloning of PCR products...........................................................................................19
2.4.1.8 Ligation......................................................................................................................19
2.4.1.9 DNA sequencing........................................................................................................20
2.4.1.10 Southern Blot analysis ...............................................................................................20
2.4.1.11 Preparation of total RNA from mouse tissues and cells.............................................20
2.4.1.12 Preparation of mRNA ................................................................................................21
2.4.1.13 cDNA synthesis .........................................................................................................21
2.4.1.14 Real-Time PCR..........................................................................................................21
2.4.1.14.1 Quantification of IIGP1 transcripts 21
2.4.1.14.2 Detection of A. phagocytophilum 22
2.4.1.14.3 n of C. trachomatis 23
2.4.2 Cell biology .........................................................................................................................23
2.4.2.1 ES cell culture............................................................................................................23
2.4.2.1.1 Thawing of cells 23
2.4.2.1.2 Freezing of cells 23
2.4.2.1.3 Mitomycin C treatment of EF cells 24
2.4.2.1.4 Transfection of ES cells 24
2.4.2.1.5 G418 selection (positive selection) 24
2.4.2.1.6 Gancyclovir (GANC) selection (negative selection) 25
2.4.2.1.7 ES colony picking 25
2.4.2.1.8 Freezing of 96 well plates 25
2.4.2.1.9 Thawing and expansion of clones from 96 well plates 26
2.4.2.1.10 His-TAT-NLS-Cre transduction of ES cells 26
2.4.2.1.11 Preparation of ES cells for blastocyst injection 26
2.4.2.2 FACS analysis............................................................................................................27
2.4.2.3 In vitro