Project Director: Kelley Thomas

A collection of full-length cDNAs is an important resource for functional genomics and for determining intron-exon boundaries within gene sequences. Six D. pulex cDNA libraries are under construction, using extracted RNA from an isoclonal line exposed to a variety of ecologically relevant conditions - hypoxia, starvation, exposure to chaoborus, fish exposure, bacteria and heavy metals - attempting to isolate a maximum diversity of DNA transcripts. These libraries are used to generate over 10,000 unique expressed sequence tags (ESTs), corresponding to ~2/3 of the predicted protein-coding genes in the Daphnia genome. The libraries are arrayed into 384-well plates and onto nylon membranes for rapid screening by PCR or hybridization for non-redundant clones and for the efficient capture of genes. Following the completions of EST sequencing projects and the subsequent annotation, the libraries are re-organized into 3 categories representing a set of unique genes that likely encode complete ORFs, a set of clones that may represent alternative transcripts, and a final set of clones that may be defective. Ultimately, a Daphnia Gene Collection will be available for distribution.

Report: Pilot project for the production of full-length cDNA libraries.

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