Development of the morpholino gene knockdown technique in Fundulus heteroclitus: a tool for studying molecular mechanisms in an established environmental model.

Abstract

A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in beta-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.

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Published Version (Please cite this version)

10.1016/j.aquatox.2008.02.010

Publication Info

Matson, Cole W, Bryan W Clark, Matthew J Jenny, Carrie R Fleming, Mark E Hahn and Richard T Di Giulio (2008). Development of the morpholino gene knockdown technique in Fundulus heteroclitus: a tool for studying molecular mechanisms in an established environmental model. Aquat Toxicol, 87(4). pp. 289–295. 10.1016/j.aquatox.2008.02.010 Retrieved from https://hdl.handle.net/10161/6969.

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Scholars@Duke

Di Giulio

Richard T. Di Giulio

Research Professor of Environmental Toxicology in the Division of Environmental Science and Policy

Dr. Di Giulio serves as Director of Duke University's Integrated Toxicology Program and the Superfund Basic Research Center.

Dr. Di Giulio's research is concerned with basic studies of mechanisms of contaminant metabolism, adaptation and toxicity, and with the development of mechanistically-based indices of exposure and toxicity that can be employed in biomonitoring. The long term goals of this research are to bridge the gap between mechanistic toxicological research and the development of useful tools for environmental assessment, and to elucidate linkages between human and ecosystem health. The bulk of Dr. Di Giulio's work employs a comparative approach with aquatic animals, particularly fishes, as models. Of particular concern are mechanisms of oxidative metabolism of aromatic hydrocarbons, mechanisms of free radical production and antioxidant defense, and mechanisms of chemical carcinogenesis, developmental perturbations and adaptations to contaminated environments by fishes.


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