Development of a Dolphin cDNA Microarray

Annalaura Mancia¹, Mats Lundqvist¹, Tracy Romano², Greg Bossart³, Patricia Fair⁴, and Gregory W. Warr¹

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Mystic Aquarium and Institute for Exploration, Mystic, CT

³ Harbor Branch Oceanographic Institution, Inc., Fort Pierce, FL.

⁴ CCEHBR, National Ocean Service, Charleston, SC

The Atlantic Bottlenose Dolphin (Tursiops truncatus) has been proposed as a sentinel species for the health of the marine environment. Dolphins, as top predators, are sensitive to the biointensification effects of marine toxins, pollutants and infectious disease agents. The aim of this project is to generate molecular tools to assess the health of wild dolphins, thereby indicating the status of the local marine environment and providing information for marine resources management. Random Expressed Sequence Tag (EST) clones have been isolated and sequenced from dolphin Peripheral Blood Leukocyte (PBL) cDNA libraries. Two cDNA libraries from known health status dolphin PBL have been generated including an IL-2 and an LPS-stimulated cDNA library, respectively biased towards T and B-cell gene expression. From the two cDNA-libraries 24,000 ESTs were collected and a total of 2200 unigenes have been sequenced and annotated (www.marinegenomics.org). Moreover, genes known to be important in the innate and adaptive immune responses of terrestrial mammals and in responses to stress and contaminant exposure have been targeted for cloning using PCR-based techniques. A total of 62 dolphin genes of known stress or immune function have been cloned by targeted PCR. The 2200 unigenes from the EST collection and the 62 immune-function targeted genes, together with other genes randomly selected without sequencing from the cDNA libraries, have been amplified and used to construct a cDNA microarray representing 3700 dolphin genes in duplicate for a total of 7400. The first set of microarray data were obtained using captive dolphin PBL RNA as probe. Results show the reproducibility of the hybridization. The slide/slide variability and the experimental variability have been validated as well. The cDNA dolphin array will be used to analyze PBL RNA from captive and wild dolphins of known health status with the aim to optimize the cDNA microarray as a sensitive and informative tool for dolphin health assessment.

This work was supported by awards from the Office of Naval Research (#N00014-02-1-0386) and from NOAA (contract #WC133C04CN0012). Studies on wild dolphins were conducted under Scientific Research Permit # 998-1678-00 from the National Marine Fisheries Service.