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Tag der mündlichen Prüfung: 29.01.2002 Contents I CONTENTS 8 2.1. Chemicals 8 2.2. Materials 10 2.3. Solutions and buffers 10 2.4. Sterilization of solutions and equipments 14 2.5. Media, antibiotics and agar-plates 15 2.5.1.1 Media for bacteria 15 2.5.1.2 Cell culture media 15 2.5.2 Antibiotics Contents II 2.6. Bacterial strains, vectors, oligonucleotide, libraries, cell lines, animals and data bases 17 2.6.1 Bacterial strains 17 2.6.2 Vectors 2.6.3 Synthetic oligonucleotide primers 2.6.4 Libraries 2.6.5 Eukaryotic cell lines 18 2.6.6 Animals 2.6.7 Data bases 2.7. Molecular biological methods 2.7.1 Isolation of nucleic acids 20 2.7.1.1 Isolation of genomic DNA from tissue samples 2.7.1.2 Isolation of total RNA from tissue 20 2.7.1.3 Isolation of poly(A)-enriched RNA 2.7.1.4 Isolation of plasmid DNA 2.7.1.4.1 Small-scale isolation of plasmid DNA 21 2.7.1.4.2 Large-scale isolation of plasmid DNA 21 2.7.1.4.3 Isolation of DNA fragments from agarose gels 2.7.1.4.4 Isolation of DNA fragments from acrylamide gels 22 2.7.1.4.5 Determination of nucleic acid concentration 2.7.2 Enzymatic modifications of DNA 2.7.2.1 Restriction of DNA 2.7.2.2 Dephosphorylation of plasmid-DNA 2.7.2.3 Ligation of DNA fragments 2.7.2.4 Transformation of bacteria 24 2.7.2.5 TA-Cloning 2.7.2.6 Filling-up reaction 25 2.7.3 Gel electrophoresis 2.7.3.1 Agarose gel electrophoresis of DNA 2.7.3.2 Agarose gel electrophoresis of RNA Contents III 2.7.3.3 DNA / RNA length standards 2.7.4 Blotting techniques 27 2.7.4.1 Dot blotting of DNA to nitrocellulose filters 27 2.7.4.2 Southern blotting of DNA to nitrocellulose filters 27 2.7.4.3 Northern blotting of RNA to nitrocellulose filters 28 2.7.5 Labelling of nucleic acids 2.7.5.1 "Random Prime" method for generation of 32 P labelled DNA 28 2.7.5.2 Hybridization of nucleic acids 2.7.5.3 Hybridization of RZPD filters 29 2.7.5.4 Chromosomal localization 2.7.6 Non-Radioactive dye terminator cycle sequencing 2.7.7 Methods of the "polymerase chain reaction" (PCR) 30 2.7.7.1 PCR of plasmid-DNA 2.7.7.2 Reverse transcriptase PCR (RT-PCR) 2.8. Cell biological methods 32 2.8.1 Transient transfection 32 2.8.2 Harvesting of the cells 32 2.8.3 Luciferase assay 2.8.4 ß-Gal assay 33 2.8.5 CAT-ELISA 33 2.8.6 Cellular localization of fusion proteins 34 2.8.7 Metaphase Arrest 2.9. Procedure for making GCAP-LTA transgenic mice using zygote injection 2.9.1 Preparation of the DNA for the microinjection RESULTS GCAP-LTA transgenic miceContents IV 3.1.1 Analysis of the human GCAP promoter region (in vitro) 36 3.1.1.1 Analysis of a 1.7 kb promoter fragment in seminoma (H12.1) and non-seminoma (1411HP) cell lines 36 3.1.1.2 Analysis of a 0.2 kb GCAP promoter fragment in different cell lines 37 3.1.1.3 Analysis of a 150-, 100-and 50 bp GCAP promoter fragment in a human embryonic carcinoma cell line (Tera-1) 40 3.1.2 Generation of GCAP-LTA transgenic mice 3.1.2.1 GCAP-LTA construct 3.1.2.2 Generation of transgenic mice 3.1.2.3 Genomic integration of the transgene 3.1.2.3.1 Analysis of transgenic founder mice (F0) generation 43 3.1...