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Investigation of Cellular Hyperosmotic Stress Response Pathways using CRISPR in Fishes
Author(s) -
Hamar Jens Carlton,
Kültz Dietmar
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb440
Subject(s) - crispr , biology , osmotic shock , microbiology and biotechnology , genome editing , cas9 , osmotic concentration , computational biology , gene , genetics , biochemistry
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 life history of many fish species includes periods of hyperosmotic stress. Two primary physiological responses a fish can employ to these conditions are systemic, attempting to maintain internal osmotic homeostasis, or cellular by adjusting the intracellular environment to the new osmotic gradient. Cell cultures can serve as an efficient model to study these pathways as seen in a cell line derived from O. mossambicus brain tissue (OmB) that retains much of the osmotically induced proteome phenotypes observed in adult tissues of this animal. Furthermore, cell culture models can facilitate these studies by providing an enhanced means to 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. The objective of these experiments is to optimize strategies to implement CRISPR/Cas9 targeting of relevant previously identified genes in OmB cells to help decipher cellular hyperosmotic stress response pathways. In this work using genetically modified OmB cells, we have shown CRISPR/Cas9 gene targeting to be successful in this cell line through targeting and reduction of transgenic enhanced green fluorescent protein (EGFP) expression and established a puromycin selection system through stable integration of puromycin resistance. Additionally, this work has demonstrated multiple interspecies promoters, both fish and mammalian, to be suitable for ectopic gene expression in fish cells that can be used in future applications. The results indicate CRISPR/Cas9 has the potential to be a proficient means to manipulate osmotic stress response relevant genes in the OmB cell model. Support or Funding Information Supported by NSF award 1656371 and BARD award IS‐4800‐15 R. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .