DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

105. Defining Complex Genetic Pathways with Gene-Expression Microarrays

M. J. Doktycz¹, B. H. Jones², C. T. Culiat², P. R. Hoyt¹, B. W. Harker¹, R. E. Barry⁴, D. D. Schmoyer³, S. Petrov³, E. M. Rinchik^2,5, K. L. Beattie¹, J. R. Snoddy³, and E. J. Michaud²

¹Biochemistry and Biophysics Section, ²Mammalian Genetics and Development Section, and ³Computational Biosciences Section, Life Sciences Division, and ⁴Robotics and Process Systems Division, Oak Ridge National Laboratory, P.O. Box 2009, Oak Ridge, TN 37831 and ⁵Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, TN 37996

okz@ornl.gov

A primary goal of functional genomics is to understand the molecular mechanisms underlying complex interactions among genetically controlled biochemical pathways and the effects of environmental exposures and aging. The complete DNA sequences of the human and mouse genomes will soon be available, including the sequences of the estimated 100,000 genes present in each of these mammals. Even now there are over 893,000 mouse expressed sequence tags (ESTs) present in databases. The availability of these EST reagents, combined with recent advances in analytical technologies and bioinformatics tools are making a dramatic impact on our comprehension of complex genetic pathways. We are exploiting these EST reagents for determining the components of genetic pathways in a single organ system, the skin. Gene-expression profiles are being determined for anonymous skin ESTs, as well as ESTs from genes with known roles in skin development, differentiation, apoptosis, DNA repair, cancer, pigmentation, and skin and hair morphology. Gene expression is being examined during normal growth and differentiation processes, and compared to expression patterns elicited in response to genetic mutations or environmental exposures. To this end, we are combining three areas of expertise at ORNL (i.e., mouse molecular genetics, analytical technologies and instrumentation, and bioinformatics) to develop an integrated-systems approach for defining gene function in genetic networks. Custom instruments, combining reagent-jets with precision movement stages, have been developed for the high throughput production of high-density microarrays. Automated procedures have been developed using commercial liquid handling systems for the preparation of tissue-specific cDNA probes, and for the parallel processing of 96 cell or tissue samples into fluorescently-labeled cDNA targets for hybridization to microarrays. Integration of these various instruments, tissue samples, cDNA clones, microarrays, and expression data will be accomplished with the aid of several inter-operating bioinformatics tools. Three bioinformation-system modules are being developed: (1) to track mice, tissues, and molecular samples; (2) to analyze the results of gene-expression arrays; and (3) to perform biologically meaningful reduction of data (e.g., by cluster analysis) and linking of the expression results to other databases containing structural and functional information (see abstract by Snoddy et al.). An important goal of this project is to make these data available to the scientific community through the web. These efforts complement the chromosome-region mutagenesis program at ORNL (see abstracts by Johnson et al., Michaud et al., and Rinchik et al.) by developing an integrated, systems-biological approach to analyzing complex multigenic traits in mice.