|
|
Component 3 is led by Dr. Pao-Tien Chuang, Assistant Professor of Biochemistry and Biophysics at UCSF. Dr. Chuang is assisted by Dr. Didier Stainier, Assistant Professor of Biochemistry and Biophysics at UCSF. Component 3 is obtaining EST clones corresponding to genes inactivated in our gene-trap studies and generating probes for in situ hybridization studies. When we identify a mouse gene that appears particularly interesting (e.g., a highly restricted pattern of expression in the heart or lung during embryonic development), the pattern of expression of the zebrafish orthologue is defined to assess evolutionary conservation of gene-expression patterns.
Recent progress includes the purchase and installation of equipment necessary to fulfill the aims outlined above. We have also performed and analyzed in situ hybridizations of IMAGE clones corresponding to genes that have been trapped by Component 1. Equipment purchased includes a Leica TP1050 (tissue processor), EG1160 (tissue embedding machine), CM3050 S (cryostat), Autostainer XL, Robbins Scientific Gemini water bath, Nikon E1000 microscope with Spot RT digital camera/PC computer, and Macintosh G4 computer. All equipment is used to process tissues, semi-automate the in situ hybridization procedure, and to capture and process the images obtained from these in situ hybridizations.
Additionally, we have made many important accomplishments toward fulfilling the aims outlined in Component 3. We have generated high-quality and consistent results with our optimized Digoxigenin In-Situ Hybridization protocol. We have performed multiple experiments using the Digoxigenin, non-isotopic, in-situ hybridization method, which we have found excellent for screening through several probes quickly. A total of 380 Image Clones were purchased from Research Genetics. Using this large-scale in situ technique we have analyzed 126 unique genes, and we are currently in the process of analyzing the latest subset, 164 genes. We have already purified and sequenced DNA from these 164 genes, and we are currently generating Digoxigenin-labeled probes and performing in-situ hybridizations. Each gene is analyzed on two tissues from different developmental stages, either 11.5, 12.5, 13.5 or 18.5 days post coitus (dpc), and on adult lung tissues, generated by Component 6, that have been treated with either bleomycin or ovalbumin to mimic lung fibrosis and asthma respectively. We have obtained satisfying in-situ results from the initial 126 genes, and all of the in-situ images have been sent to Component 2 for posting on the BayGenomics website (you can browse the list of images). Unfortunately, the sequences from the remaining 90 clones did not match their corresponding IMAGE clone sequence posted in GenBank nor any gene trapped lines in the BayGenomics database. Thus, Research Genetics has sent us a shockingly high number of clones that do not match the ones that we actually ordered.