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Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR c...

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Autores principales: Notch, Emily G., Shaw, Joseph R., Coutermarsh, Bonita A., Dzioba, Marisa, Stanton, Bruce A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245286/
https://www.ncbi.nlm.nih.gov/pubmed/22216285
http://dx.doi.org/10.1371/journal.pone.0029462
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author Notch, Emily G.
Shaw, Joseph R.
Coutermarsh, Bonita A.
Dzioba, Marisa
Stanton, Bruce A.
author_facet Notch, Emily G.
Shaw, Joseph R.
Coutermarsh, Bonita A.
Dzioba, Marisa
Stanton, Bruce A.
author_sort Notch, Emily G.
collection PubMed
description The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism.
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spelling pubmed-32452862012-01-03 Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation Notch, Emily G. Shaw, Joseph R. Coutermarsh, Bonita A. Dzioba, Marisa Stanton, Bruce A. PLoS One Research Article The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism. Public Library of Science 2011-12-22 /pmc/articles/PMC3245286/ /pubmed/22216285 http://dx.doi.org/10.1371/journal.pone.0029462 Text en Notch et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Notch, Emily G.
Shaw, Joseph R.
Coutermarsh, Bonita A.
Dzioba, Marisa
Stanton, Bruce A.
Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
title Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
title_full Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
title_fullStr Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
title_full_unstemmed Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
title_short Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation
title_sort morpholino gene knockdown in adult fundulus heteroclitus: role of sgk1 in seawater acclimation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245286/
https://www.ncbi.nlm.nih.gov/pubmed/22216285
http://dx.doi.org/10.1371/journal.pone.0029462
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