Seminar Details

Joe Shaw, Dartmouth College
Using microarrrays to determine metal induced gene-response in Daphnia pulex

November 14, 2004 to November 18, 2004
Portland, Oregon, USA

Abstract:

Fourth SETAC World Congress & 25th Annual Meeting in North America

Shaw, J., Chen, C., Davey, J., Folt, C., Hamilton, J., Colbourne, J., Dartmouth College, Hanover, NH, USA2 Dartmouth Medical School, Hanover, NH, USA & Indiana University, Bloomington, IN, USA

Investigations were conducted to determine the genomic response of Daphnia pulex to arsenic and cadmium. These metals were selected for study in part because of their differences in toxic mechanisms. Daphnia was selected because of its sentinel role within aquatic ecosystems and because genomic resources for this model species are now available. The current list of genomic tools includes a high-quality microarray with over 3400 unidentified targets that are derived from full length cDNA libraries. Based on random sequencing of 600 cDNAs, we estimated that 35% of the arrayed targets are redundant, suggesting that the array contains 2210 unique genes. These targets were robotically spotted in tandem, along with controls, on glass slides. To discover differential gene expression patterns under metal stress, Daphnia were acutely (48-h) exposed to incipient concentrations (LC10) of each metal (20 g Cd/L and 1384 g As/L). RNA was isolated from metal exposed animals (both treatments) and from their genetic clones under standard (reference) conditions. Each treatment and reference sample was labeled indirectly using aminoallyl-dNTP coupling and alternate Alexa Fluor dyes (555, 647). Our replicated experiments measured the relative amounts of gene transcripts between both conditions. Statistical analyses revealed that a small number of genes were differentially expressed in the two metal treatments (2-5%). However, arsenic and cadmium produced almost entirely different transcriptional responses as there was very little overlap (5%) between these two groups. These blind arrays allowed for identification of differentially expressed genes prior to sequencing, but required post-hoc analysis to identify genes. As such, these identified probes are being sequenced and differential responses validated by real-time (quantitative) PCR. Future studies will focus on chronic/sub-lethal exposures, linking genomic response and demographic outcome with the ultimate goal of establishing predictive biomarkers that could be employed to determine the susceptibility of natural populations to metals.