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Generation and Characterization of Spred-2 Knockout Mice

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Generation and Characterization of Spred-2 Knockout Mice Generation and Characterization of Spred-2 Knockout Mice Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Dr. med. Karin Andrea Bundschu (geb. Geis) aus Frankfurt/Main Würzburg 2005 Eingereicht am: .....…………………………………………………………….. Mitglieder der Promotionskommission: Vorsitzender: ...……………………………………………………………….. Gutachter: Prof. Dr. U. Walter Gutachter: Prof. Dr. G. Krohne Betreuer: Dr. Kai Schuh Tag des Promotionskolloquiums: …………………………………………… Doktorurkunde ausgehändigt am: …………………………………………… The present study was performed under supervision of Dr. Kai Schuh in the group of Prof. Dr. med. Ulrich Walter in the Institute of Clinical Biochemistry and Pathobiochemistry at the Julius-Maximilians-University of Würzburg. The dissertation was part of the MD/PhD program, organized by the IZKF of the University of Würzburg. Declaration: Hereby, I declare that the submitted dissertation was completed by myself and no others and that I have not used any sources or materials other than those enclosed. Moreover, I declare that the following dissertation has not been submitted further in this form or any other form and has not been used for obtaining any other equivalent qualification in any other organization. Additionally, other than this degree I have not applied or will attempt to apply for any other degree or qualification in relation to this work. Würzburg, (Dr. med. Karin Andrea Bundschu) In love for my husband Christoph and our son Sebastian Table of Contents TABLE OF CONTENTS 1. SUMMARY.......................................................................................................... 5 2. ZUSAMMENFASSUNG ...................................................................................... 7 3. INTRODUCTION................................................................................................. 9 3.1. EVH-1 (ENA/VASP HOMOLOGY-1) DOMAIN PROTEINS....................................... 9 3.1.1. Ena/VASP proteins ................................................................................... 9 3.1.2. WASP proteins ....................................................................................... 10 3.1.3. Homer/Vesl proteins ............................................................................... 10 3.2. SPROUTY PROTEINS...................................................................................... 11 3.3. SPRED PROTEINS.......................................................................................... 13 3.4. THE MAP KINASE SIGNALING PATHWAY .......................................................... 15 3.5. KNOCKOUT STRATEGIES IN MICE .................................................................... 16 3.6. BONE GROWTH AND FGF-MEDIATED ENDOCHONDRAL OSSIFICATION ................. 19 3.7. AIM OF THE WORK ......................................................................................... 21 4. MATERIAL AND METHODS ............................................................................ 22 4.1. MOLECULAR-BIOLOGICAL METHODS................................................................ 22 4.1.1. Database analyzes of the mouse genome.............................................. 22 4.1.2. Isolation of plasmid DNA ........................................................................ 22 4.1.3. Isolation of genomic DNA of mouse tails and ES cells ........................... 22 4.1.4. DNA gel electrophoresis ......................................................................... 22 4.1.5. DNA purification of agarose gels ............................................................ 22 4.1.6. Purification of PCR products................................................................... 23 4.1.7. Cutting DNA by endonucleases.............................................................. 23 4.1.8. Dephosphorylation of DNA 5’ overhangs................................................ 23 4.1.9. Ligation/cloning of DNA fragments in a plasmid vector........................... 23 4.1.10. T/A-cloning.......................................................................................... 23 4.1.11. Generation of competent bacterias ..................................................... 23 4.1.12. Transformation of competent bacterias............................................... 24 4.1.13. Verification of inserts........................................................................... 24 4.1.14. LA- (long and accurate) PCR .............................................................. 25 4.1.15. Sequencing (Sanger method) ............................................................. 25 4.1.16. Southern blot analyzes........................................................................ 25 1 Table of Contents 4.1.17. Cloning of Spred-1 and Spred-2 full length cDNA in TOPO2.1 ........... 26 4.1.18. Cloning of Spred-1 and Spred-2 cDNA in pcDNA3.1/Myc-His ............ 27 4.1.19. RNA extraction of mouse tissues ........................................................ 27 4.1.20. DNA removal of RNA extracts............................................................. 28 4.1.21. RT- (Reverse Transcription) PCR ....................................................... 28 4.1.22. Northern blot analyzes ........................................................................ 28 4.2. HISTOLOGICAL METHODS............................................................................... 29 4.2.1. Paraffin sections and HE staining........................................................... 29 4.2.2. Immunohistochemistry............................................................................ 29 4.2.3. X-Gal staining ......................................................................................... 30 4.2.4. Toluidine staining.................................................................................... 30 4.3. CELL CULTURE ............................................................................................. 31 4.3.1. Transfection of HEK 293 cells................................................................. 31 4.3.2. Primary mouse chondrocyte culture ....................................................... 31 4.4. PROTEIN BIOCHEMICAL METHODS ................................................................... 31 4.4.1. Protein isolation of mouse tissues .......................................................... 31 4.4.2. Western blot analyzes ............................................................................ 32 4.4.3. Coomassie-blue protein staining of SDS gels......................................... 32 4.5. GENERATION AND AFFINITY PURIFICATION OF POLYCLONAL SPRED-1 AND SPRED-2 SPECIFIC ANTIBODIES ............................................................................................... 33 4.5.1. Expression and preparation of GST-Spred-1 and GST-Spred-2 fusion proteins.............................................................................................................. 33 4.5.2. Coupling of proteins to NHS-HiTrap columns ......................................... 35 4.5.3. Immunization of rabbits........................................................................... 35 4.5.4. Affinity purification of antisera................................................................. 35 4.6. GENERATION OF SPRED-2 KNOCKOUT MICE USING A GENE TRAP MODEL ............ 37 4.6.1. Gene trapping and Southern blot analysis.............................................. 37 4.6.2. Mouse breeding ...................................................................................... 38 4.6.3. Genotyping of mice................................................................................. 38 4.7. GENERATION OF SPRED-1 AND SPRED-2 KNOCKOUT MICE USING GENE TARGETING VECTORS ................................................................................................................ 39 4.7.1. General strategy ..................................................................................... 39 4.7.2. Cloning of Spred-1 and Spred-2 gene targeting vectors......................... 40 4.7.3. Optimization of the embryonic stem (ES) cell screening PCR ................ 45 2 Table of Contents 4.7.4. Electroporation and selection of ES cells................................................ 46 4.7.5. PCR screening of ES cells for homologous recombination events ......... 46 4.7.6. Cre-recombinase treatment of ES cells and PCR screening of different Cre-recombinase events for complete and conditional knockouts..................... 47 4.8. MOUSE PHYSIOLOGY..................................................................................... 48 4.8.1. Hormone measurements in mice............................................................ 48 4.8.2. Blood glucose measurements in mice .................................................... 48 4.8.3. Blood cell counts in mice ........................................................................ 48 4.8.4. Bleeding time measurements in mice ..................................................... 48 4.8.5. Bleeding volume measurements in mice ................................................ 48 4.8.6. Heart parameter measurements in mice................................................. 49 4.8.7. Estimation of bone lengths ..................................................................... 49 4.8.8. Statistical analyzes ................................................................................. 49 4.9. ADDITIONAL MATERIALS AND EQUIPMENT........................................................
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