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Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

ABSTRACT: Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (? passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

SUBMITTER: Gardell AM

PROVIDER: S-EPMC4010420 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Gardell Alison M AM Qin Qin Q Rice Robert H RH Li Johnathan J Kültz Dietmar D

PloS one 20140505 5

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementati ...[more]

PMID: 24797371

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Project description:BACKGROUND:Osmotic stress is a widespread phenomenon in aquatic animal. The ability to cope with salinity stress and alkaline stress is quite important for the survival of aquatic species under natural conditions. Tilapia is an important commercial euryhaline fish species. What's more tilapia is a good experimental material for osmotic stress regulation research, but the molecular regulation mechanism underlying different osmotic pressure of tilapia is still unexplored. RESULTS:To elucidate the osmoregulation strategy behind its hyper salinity, alkalinity and salinity-alkalinity stress of tilapia, the transcriptomes of gills in hybrid tilapia (Oreochromis mossambicus ??×?O. urolepis hornorum ?) under salinity stress (S: 25‰), alkalinity stress(A: 4‰) and salinity-alkalinity stress (SA: S: 15‰, A: 4‰) were sequenced using deep-sequencing platform Illumina/HiSeq-2000 and differential expression genes (DEGs) were identified. A total of 1958, 1472 and 1315 upregulated and 1824, 1940 and 1735 downregulated genes (P-value <?0.05) were identified in the salt stress, alkali stress and saline-alkali stress groups, respectively, compared with those in the control group. Furthermore, Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted in the significant different expression genes. In all significant DEGs, some of the typical genes involved in osmoregulation, including carbonic anhydrase (CA), calcium/calmodulin-dependent protein kinase (CaM kinase) II (CAMK2), aquaporin-1(AQP1), sodium bicarbonate cotransporter (SLC4A4/NBC1), chloride channel 2(CLCN2), sodium/potassium/chloride transporter (SLC12A2 / NKCC1) and other osmoregulation genes were also identified. RNA-seq results were validated with quantitative real-time PCR (qPCR), the 17 random selected genes showed a consistent direction in both RNA-Seq and qPCR analysis, demonstrated that the results of RNA-seq were reliable. CONCLUSIONS:The present results would be helpful to elucidate the osmoregulation mechanism of aquatic animals adapting to saline-alkali challenge. This study provides a global overview of gene expression patterns and pathways that related to osmoregulation in hybrid tilapia, and could contribute to a better understanding of the molecular regulation mechanism in different osmotic stresses.

Dataset Information

Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Publications

Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

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