Meeting Abstract
Osmotic stress is one of the primary challenges faced by aquatic animals and their capacity to osmoregulate in response will dictate its survivability within a given set of conditions. The Mozambique Tilapia (Oreochromis mossambicus) is an ideal model organism for studying the physiology behind this response due its extreme ability to adapt to this type of stress. A cell line was derived from O. mossambicus brain tissue (OmB) that retains much of the osmotically induced proteome phenotypes observed in adult tissues of this animal. Cell cultures can facilitate these studies by providing a means to further isolate and examine individual components, such as proteins and the genes that encode them, of the mechanisms behind adaption to osmotic challenges. Valuable functional information can be obtained through observation of specific phenotypes caused by genetic manipulations of these components. CRISPR/Cas9 gene editing systems have proven to be a potent means to induce targeted genetic manipulations in cell culture from a variety of vertebrate taxa including fish. A “proof of principle” assay was performed to demonstrate function of the CRISPR/Cas9 system in the OmB cell line by targeting and knocking out transgenic expression of EGFP. Following treatment with expression plasmids encoding the required EGFP targeted CRISPR/Cas9 components, examination by fluorescence microscopy showed a marked reduction of cells with detectable EGFP fluorescence relative to controls providing visual evidence of function of the system in these cells. Current work involves adapting a plasmid based system to implement CRISPR/Cas9 targeting of relevant previously identified genes to help decipher cellular osmotic stress response mechanisms using the OmB cell line model. Funded by NSF grant IOS-1355098 and BARD grant IS-4800-15R.
