We are developing C. elegans whole genome DNA microarrays, which will be used for gene expression profiling. Software developed at the Sanger Centre by Steve Jones has been used to predict PCR primer pairs that will specifically amplify 1 kb exon-rich regions from each of the predicted 19,000 C. elegans genes in the C. elegansgenome sequence. Primers have been generously provided by Stuart Kim (Stanford, CA). The DNA microarrays will be produced following protocols originally developed in the laboratory of P. Brown (Stanford, CA) and also used by S. Kim. The microarrays will be generated by robotically gridding each of the 18,000 1kb DNA fragments onto coated glass slides. The microarrays will then be used to compare steady-state RNA populations expressed by animals with different genetic backgrounds or physiologies by differential hybridisation of cDNAs. These cDNAs will carry either a Cy3 or Cy5 fluorescent tag to be incorporated during reverse transcription. Following hybridisation, the resulting fluorescent signals will be detected by confocal laser scanning and expressed as two-colour ratios of differential expression. We plan to use the microarrays to address a series of key questions in developmental biology. For example, we plan to compile a profile of downstream targets for many of the predicted homeodomain proteins in the worm. To enable us to do this, we have been taking advantage of the C. elegansgenome sequence to design PCR primers flanking the predicted homeobox genes. RT-PCR is then used to generate cDNAs for these genes, and then the cDNA is cloned into heatshock vector pPD49.83. We plan to generate transgenic worms, co-injecting these constructs with an appropriate selectable marker. To date, 6 homeobox genes have been successfully cloned into pPD49.83. These are C17H12.9, F17A9.6, F46C8.5 (ceh-14), R08B4.2, ZC64.4 (lim-4), and ZC247.3 (lin-11). The sequences of the cDNAs for these genes in some cases revealed mispredictions of the intron-exon boundaries for the genes as given by ACeDB. We hope to obtain cDNA clones or heatshock constructs from other labs to expedite the process, and have already received several HOX gene constructs from various labs, for which we are extremely grateful. The power of DNA microarray technology is two-fold: DNA microarrays yield functional information on genes in the form of expression profiles, and they enable an entire genome to be searched in a single reaction to identify primary and secondary response genes. Quantitative data can be obtained on both up- and down-regulated genes to help decipher genetic pathways. The technology is sufficiently sensitive to detect 2-fold differences in gene expression. Finally, we plan to make this technology and the data obtained available to the members of the research community.