Most of our energy and resources have been spent on large multi-Institutional projects during this fiscal year. These include the Mammalian Gene Collection (MGC), the Brain Molecular Anatomy Project (BMAP), GenSAT, the Transcriptome Project, and the Alliance for Cell Signaling. The goal of the MGC is to clone and sequence full-length (or full-open reading frame) cDNAs corresponding to all human and mouse transcripts, and to put the sequences and DNAs in the public domain. In addition to preparing human and mouse tissues and RNAs for use by members of the MGC and Transcriptome project consortia, members of the Laboratory have taken responsibility for assembling a complete collection of human cDNAs that encode G-protein coupled receptors (GPCRs), and transferring them to the pcDNA3.1 vector so that they can readily be used for functional studies. We focused on these receptors because they are the targets of about 50% of the drugs in use today. Information about many of the members of this large family of proteins is sketchy at best, and learning more about them could result in their being selected as targets for drug development in the future. At least 4 of the receptors cloned by members of the Laboratory in the past (the three vasopressin receptors and the cannabinoid CB1 receptor) are such targets already. Since many of the 340 GPCRs (excluding odorant receptors) have been cloned by others, we have requested donations of cDNAs. Donors are acknowledged as members of the """"""""GPCR Consortium"""""""". The response to requests for clones has been gratifying, but many of the GPCR cDNAs have had to be isolated again, and their sequences validated. This has taken and continues to take a good deal of time; our collection now includes 260 sequence validated DNAs in expression vector. The BMAP and GenSAT projects have similar goals: to determine which genes are expressed in the developing and adult mouse and human nervous systems, in which cells they are expressed, and when their products are produced. We reasoned that a low-resolution answer to these questions could be obtained by using cDNA microarrays to profile gene expression in developing mouse brains, regions of the adult central nervous system, and peripheral tissues. The results of such studies are available at http://intramural.nimh.nih.gov/research/log/index2.html. In order to obtain the data shown, we found that we had to improve the methods that were available for labeling probes for microarray studies and for amplifying RNA templates. Technical development is an ongoing effort. In the last year we have developed and validated methods for fixing and immunostaining sections that should allow us to capture homogeneous populations of neurons from the brain and extract high quality RNA from them. Further work is needed to make all of the techniques involved """"""""routine"""""""" and reliable. Scientists in GenSAT laboratories will use in situ hybridization histochemistry to visualize specific mRNAs in mouse brain sections. This will allow them to identify which cells make the RNAs selected for study. The BMAP array data has allowed us to select certain transcripts (in addition to GPCRs) for special attention in the GenSAT project. These are mRNAs that have not already been mapped in detail and that seem to be expressed in a one region, at one time, or in female or male animals preferentially. Finally, it should be mentioned that we are presently supported by the NIAID to develop a rapid, sensitive, and specific array-based method for rapid detection of Category A pathogens. Work on this project has gone well.
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