Worm Breeder's Gazette 15(3): 10 (June 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Dept. of Developmental Biology, Stanford U., Stanford, CA 94305.
We are currently adapting a cDNA microarray-based method for monitoring differential gene expression for use in worms. DNA microarrays consist of PCR fragments that have been robotically deposited onto treated microscope slides. To analyze differential gene expression between two strains, cDNA probes are made from each strain using fluorescently-labeled nucleotides. Two probes, each labeled with a different fluorescent dye, are simultaneously hybridized to a microarray. The microarray is scanned with a scanning confocal-laser microscope and the emission intensity of each fluorescent dye at every location on the array is measured. By normalizing to total hybridization signals from each probe, relative differences in hybridization intensity can be determined, permitting us to calculate gene expression differences. For example, cDNA probe prepared from wild-type and a mutant might be labeled green and red, respectively. Green spots would represent genes that are expressed less in the mutant relative to wild-type. Conversely, red spots would represent genes that are more highly expressed in the mutant relative to wild-type. Genes expressed at similar levels would appear yellow. One powerful application of the microarray is to identify transcriptional differences in a mutant versus wild-type. In particular, genes that act redundantly with other genes could be identified. A limitation of the microarray-based approach for worms is that isolation of RNA must occur from whole worms. Thus, the microarray may not be sensitive enough to detect differences in transcripts that are only present in a few cells. Alternatively, constitutive gene expression in tissues outside of the cells of interest may obscure relevant gene expression differences. Currently, we have produced a 1264 element array, of which 1152 elements are PCR products from a library of random C. elegans ESTs (generously provided by Yuji Kohara at National Institute of Genetics, Japan). In collaboration with Jeremy Nance and Sam Ward, we have used the DNA microarrays to identify potential sperm and oocyte enriched genes. As described in an accompanying Gazette article, the sperm enriched genes identified in this experiment included a number of major sperm protein genes, and the oocyte enriched genes identified several meiosis and maternally expressed genes. We have begun to identify genes regulated by the MAP kinase signaling pathway in vulval development. We welcome collaborations with any persons interested in applying microarray technology to their studies.