Clark, Karl J.

Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21 st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish ( Danio rerio ) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav /cct8 and hbog /gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning.

Abstract Morpholino oligonucleotides are the most common anti-sense “knockdown” technique used in zebrafish ( Danio rerio ). This review discusses common practices for the design, preparation, and deployment of morpholinos in this vertebrate model system. Off-targeting effects of morpholinos are discussed as well as method to minimize this potentially confounding variable via co-injection of a tP53-targeting morpholino. Finally, new uses of morpholinos are summarized and contextualized with respect to the complementary, DNA-based knockout technologies recently developed for zebrafish.