Evaluation of the coxsackievirus and adenovirus receptor (CAR) as a therapeutic target in cardiac disease [Elektronische Ressource] / von Chen Chen

Chen Chen

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2009
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 12 Mo

Extrait

Evaluation of the Coxsackievirus
and Adenovirus Receptor (CAR) as a
therapeutic target in cardiac disease

Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der

Mathematisch-Naturwissenschaftliche Fakultät I
der Humboldt-Universität zu Berlin

von
M. Sc. Chen Chen
Geb. am 15.11.1979, Zhejiang, China

Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön
Gutachter: 1. Prof. Dr. Michael Gotthardt
2. Prof. Dr. Harald Saumweber
3. Prof. Dr. Michael Bader
Datum der Einreichung: 27.03.2009
Datum der Promotion: 09.07.2009


Table of Contents
Table of Contents ................................................................................................. i 
Zusammenfassung ............................................................................................... v 
Abstract .............................................................................................................. vii 
1  Introduction .................................................................................................... 1 
1.1  The Coxsackievirus and Adenovirus Receptor (CAR) ............................. 1 
1.1.1  CAR gene and splice isoforms ...................................................... 2 
1.1.2  CAR protein domain structure and function ................................. 3 
1.1.3  Evolutionary conservation ............................................................. 7 
1.1.4  CAR expression ............................................................................. 8 
1.1.5  CAR knockout models ................................................................ 15 
1.2  CAR is a tight junction protein ............................................................... 17 
1.3  CAR mediates virus uptake ..................................................................... 19 
1.4  Aim of the study ...................................................................................... 22 
2  Materials ....................................................................................................... 24 
2.1  Chemicals and reagents ........................................................................... 24 
2.2  Kits ..................................................................................................... 24 
2.3  Solutions .................................................................................................. 24 
2.4  Cell culture media ................................................................................... 25 
2.5  Antibodies ............................................................................................... 26 
2.6  Oligonucleotides ..................................................................................... 26 
2.7  Appliances28 
2.8  Software29 
2.9  Animals ................................................................................................... 30 
3  Methods ......................................................................................................... 31 
3.1  Molecular biology methods ..................................................................... 31 
3.1.1  DNA preparation ......................................................................... 31 
3.1.2  PCR based genotyping ................................................................ 31 
3.1.3  Agarose gel electrophoresis ......................................................... 32 
3.1.4  Determination of nucleic acid concentration ............................... 33 
3.1.5  Generation of the targeting vector ............................................... 33 
i

3.1.6  DNA Sequencing ......................................................................... 33 
3.1.7  Southern blot with genomic DNA ............................................... 34 
3.1.8  Total RNA isolation and purification .......................................... 35 
3.1.9  Formaldehyde agarose gel electrophoresis .................................. 36 
3.1.10  Synthesis of cDNA ...................................................................... 36 
3.1.11  Real-time PCR ............................................................................. 37 
3.1.12  Microarray analysis37 
3.2  Cell biology methods ............................................................................... 38 
3.2.1  Isolation of primary mouse embryonic cardiomyocytes .............. 38 
3.2.2  Isolation of embryonic fibroblasts ............................................... 39 
3.2.3  Isolation of epithelial cells from yolk sac .................................... 39 
3.2.4  Isolation of adult cardiomyocytes ................................................ 40 
3.2.5  Preparation of cardiac muscle slices and dye coupling studies ... 41 
3.3  Biochemical methods ............................................................................... 42 
3.3.1  Preparation of total protein extract and quantification ................ 42 
3.3.2  SDS-polyacrylamide electrophoresis (SDS-PAGE) .................... 43 
3.3.3  Western blotting ........................................................................... 44 
3.4  Animal procedures ................................................................................... 45 
3.4.1  Setting up timed matings and dissection ...................................... 45 
3.4.2  Preparation of paraffin sections ................................................... 45 
3.4.3  Immunoperoxidase staining ......................................................... 45 
3.4.4  H&E staining ............................................................................... 46 
3.4.5  PAS staining ................................................................................. 47 
3.4.6  Trichrome staining ....................................................................... 47 
3.4.7  Immunoperoxidase staining47 
3.4.8  Preparation of cryosections .......................................................... 48 
3.4.9  Immunofluorescence staining ...................................................... 48 
3.4.10  Tamoxifen injection ..................................................................... 49 
3.4.11  Virus infection ............................................................................. 49 
3.4.12  In situ hybridization and quantification of CVB3 infection ........ 50 
3.5  Confocal microscopy ............................................................................... 50 
3.5.1  3D reconstruction ......................................................................... 51 
3.6  Surgical procedures ................................................................................. 51 
3.6.1  Hemodynamic measurements ...................................................... 51 
ii

3.6.2  Surface ECG and in vivo electrophysiology studies ................... 51 
3.6.3  Arteriovenous (AV) shunt ........................................................... 52 
3.7  Statistics .................................................................................................. 52 
4  Results ........................................................................................................... 53 
4.1  Generation of the coxsackievirus and adenovirus receptor (CAR)
conventional and tissue specific conditional knockout model ................ 53 
4.2  CAR is required for the early embryonic development .......................... 56 
4.2.1  Deletion of CAR causes embryonic lethality at midgestation .... 56 
4.2.2  CAR deficiency causes heart and vessel malformation .............. 57 
4.2.3  Myofibril disorganization in knockout embryonic cardiac cells . 58 
4.2.4  The structure of ependymal cells in the brain was disrupted ...... 60 
4.2.5  Expression levels of connexins and apolipoproteins are altered in
the embryonic CAR KO heart ..................................................... 61 
4.3  Cardiac deletion of the coxsackievirus and adenovirus receptor abolishes
CVB3 infection and prevents myocarditis in vivo .................................. 63 
4.3.1  Tamoxifen predisposes mice to lethal CVB3 induced pancreatitis63 
4.3.2  Cardiac CVB3 infection can be abolished by eliminating CAR . 65 
4.3.3  Loss of CAR prevents viral myocarditis ..................................... 66 
4.3.4  Cardiac function is preserved in CAR deficient mice after CVB3
infection .................