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Susceptibility to Liver Carcinogenesis Is Increased in a Mouse Model of Conditional E Cadherin Deficiency

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Susceptibility to Liver Carcinogenesis Is Increased in a Mouse Model of Conditional E Cadherin Deficiency Aus dem Veterinärwissenschaftlichen Department der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Arbeit angefertigt unter der Leitung von PD Dr. Marlon R. Schneider Susceptibility to liver carcinogenesis is increased in a mouse model of conditional E-cadherin deficiency Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München von Felix Nico Hiltwein aus Berlin-Zehlendorf München 2013 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Joachim Braun Berichterstatter: Priv.-Doz. Dr. Marlon Schneider Korreferent: Univ.-Prof. Dr. Johannes Hirschberger Tag der Promotion: 20. Juli 2013 Dedicated to all those closest & dearest to me, be it my biological, sociological or emotional family. “The best-laid schemes o' mice an' men / Gang aft agley” ("The best-laid plans of mice and men / Often go awry") Robert Burns, 1785 Table of contents V TABLE OF CONTENTS I. INTRODUCTION ...................................................................................... 1 II. REVIEW OF THE LITERATURE .......................................................... 3 1. Genetically modified mice .........................................................................3 1.1. Functional analysis of the genome (in any organism)..................................3 1.2. Why mice?....................................................................................................4 1.3. Creation of genetically modified mice .........................................................5 1.4. Conditional knockout (Cre/loxP) .................................................................8 1.5. Analyzing the phenotype of genetically manipulated animals ...................11 2. E-Cadherin ................................................................................................12 2.1. Cadherins in general ...................................................................................12 2.2. E-Cadherin structure, expression and relevance ........................................14 2.3. Connection of E-cadherin with tumors ......................................................16 3. Hepatocellular carcinoma (HCC) ...........................................................18 3.1. HCC in humans ..........................................................................................19 3.2. HCC in mice (induced by diethylnitrosamine) ..........................................20 III. ANIMALS, MATERIALS AND METHODS ........................................ 22 1. Animals ......................................................................................................22 1.1. Cdh1 conditional knockout mice................................................................22 1.2. Alb-Cre transgenic mice.............................................................................22 1.3. Mouse breeding procedure .........................................................................22 1.4. Animal maintenance ...................................................................................23 1.5. Approval of animal testing and ethical guidelines .....................................23 2. Mouse genotyping .....................................................................................23 2.1. Tissue collection .........................................................................................24 2.2. Extraction of DNA from mouse tail tips ....................................................25 2.3. Principle of the polymerase chain reaction (PCR) .....................................25 2.4. Assay procedure and PCR protocols ..........................................................26 3. Basic overview of mouse sections ............................................................27 4. Assessment of E-cadherin loss on protein-level .....................................28 4.1. Collection of liver samples .........................................................................28 VI Table of contents 4.2. Preparation of liver samples ...................................................................... 28 4.3. Electrophoresis and blotting ...................................................................... 29 4.4. Detection of protein ................................................................................... 31 5. Evaluation of gene expression at the RNA level.................................... 31 5.1. Collection of liver samples for RNA analysis ........................................... 31 5.2. RNA expression analysis using Agilent Microarray ................................. 32 5.3. cDNA synthesis for qRT-PCR ................................................................... 32 5.4. Quantitative RT-PCR ................................................................................. 33 6. Analysis of body weight development and organ weight ..................... 34 6.1. Long-term body weight development ........................................................ 34 6.2. Body and organ weight at specific time points .......................................... 34 7. Evaluation of serum parameters ............................................................ 34 7.1. Blood collection ......................................................................................... 34 7.2. Serum preparation ...................................................................................... 35 7.3. Clinical chemistry ...................................................................................... 35 8. Induction of tumors through use of diethylnitrosamine (DEN) .......... 35 8.1. Preparation of DEN and dosage................................................................. 36 8.2. Intraperitoneal (i.p.) application ................................................................ 36 8.3. Safety measures ......................................................................................... 36 9. Section of mice with tumors .................................................................... 37 9.1. Liver and body weight ............................................................................... 37 9.2. Other organ weights ................................................................................... 37 9.3. Count of tumor lesions ............................................................................... 38 9.4. Preparation of tumor samples for RNA analysis, histology and cell culture . ................................................................................................................... 38 10. Establishment of long term cell culture from tumors .......................... 39 10.1. Preparation of collagenase II ..................................................................... 39 10.2. Mincing of tumors and destruction of the organ/tumor structure .............. 39 10.3. Erythrocyte lyses........................................................................................ 40 10.4. Re-suspension in cell culture dishes .......................................................... 40 10.5. Maintenance and splitting of cell lines ...................................................... 41 10.6. PCR of cell culture cells ............................................................................ 41 10.7. Conservation of cell-lines for future studies .............................................. 41 Table of contents VII 11. Histological analysis .................................................................................41 11.1. Hematoxilin and eosin (H&E) staining ......................................................42 11.2. Immunohistochemistry (IHC) ....................................................................42 12. Histological evaluation of HCC ...............................................................43 12.1. Histological quantification of tumors in the liver ......................................43 12.2. Histological evaluation of tumors in the lung ............................................44 13. Statistical analysis ....................................................................................44 14. Primer sequences ......................................................................................45 14.1. Primers for genomic DNA amplification ...................................................45 14.2. Primers for cDNA amplification ................................................................45 15. Materials ...................................................................................................46 15.1. Machines ....................................................................................................46 15.2. Consumables ..............................................................................................48 15.3. Chemicals ...................................................................................................49 15.4. Drugs (used on animals) .............................................................................53 15.5. Microarray technology ...............................................................................53 15.6. Software .....................................................................................................53 15.7. Animals ......................................................................................................54 IV. RESULTS .................................................................................................. 55 1. Spontaneous
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