This proposal responds to the Program """"""""Development of Non-mammalian Models and Related Biological Materials for Research."""""""" Specifically, the program announcement mentions """"""""Species such as fishes...and cell lines and cell culture models for non-mammalian animals where culture systems are not well developed but would be widely used."""""""" We propose to develop cell lines from marine elasmobranchs (sharks and skates), animals that are broadly exploited in biomedical research as models in physiology, endocrinology, toxicology and genomics. Currently there are no homologous in vitro systems by which investigators using these models can test hypotheses or confirm predictions at a molecular level, and application of new technologies is needed. The premise driving this project is that in vitro culture of specialized cell types can be accomplished by taking non-conventional approaches, including removal or reduction of serurnas a medium component and replacement with cell type-specific supplements, such as peptide growth factors. We have used this approach to generate cell lines from zebrafish, pufferfish (fugu), and other fish species. This work will take advantage of the unique resources of the Mount Desert Island Biological Laboratory, a research facility dedicated to the study of marine organisms to discover biological mechanisms of significance to human health, and world center for the use of the dogfish shark (Squalus acanthias) and little skate (Raja erinacea) as research models. We propose to develop differentiation-competent, continuously proliferating cell lines from (1) liver, brain, kidney and salt (rectal) gland of the dogfish shark, and (2) liver and embryo of the little skate. We also propose to (3) develop systems for DNA transfection and plasmid-derived gene expression in these cell lines. Success will provide a platform for original and innovative experiments, accelerating the development of concepts driving multiple areas of biomedical research addressed by the large number of investigators using these models.
