Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

doi:10.1371/journal.pone.0095919

Cited by 44 publications

(50 citation statements)

References 84 publications

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“…S5 C and D). These results are entirely consistent with our previous data showing that tilapia IMPA1.1 is highly induced, whereas IMPA1.2 is marginally induced in both OmB cells and brain of intact O. mossambicus exposed to salinity stress (38,43). Moreover, in contrast to IMPA1.1 mRNA, the abundance of IMPA1.3 mRNA is not altered in any of the four O. niloticus tissues tested (gill, kidney, fin, and intestine) when salinity increases from fresh water (FW) to 50% or 100% seawater (SW) (53).…”

Section: Discussionsupporting

confidence: 93%

“…This pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol and is highly salinity-induced in multiple tissues of O. mossambicus, including gill epithelium, brain, heart, larval epidermis, and OmB cells (38,(41)(42)(43)51). Induction of these two genes is also evident in other euryhaline fish exposed to acute salinity stress.…”

Section: Discussionmentioning

confidence: 99%

“…To enable enhancer trap reporter assays, we previously established immortalized O. mossambicus cell lines that are highly tolerant of acute hyperosmolality up to 700 mOsm/kg (38). Establishing cell lines was crucial because the isolation and identification of the specifies-specific transcriptional machinery and trans-acting factor that have coevolved with the salinity-induced CRE would be compromised in a heterologous background.…”

Section: Discussionmentioning

confidence: 99%

“…We recently established several immortalized O. mossambicus cell lines to render enhancer trap reporter assays feasible for euryhaline fish (38). Cultured cells are much more amenable to mechanistic and causal dissection of environmental stress responses than tissues of complex and long-lived organisms analyzed in situ (39,40).…”

Section: Significancementioning

confidence: 99%

“…Therefore, if the cellular and biochemical phenotypes observed in the tissues of whole organisms are reproducible in cell culture, their mechanistic basis can be revealed using cell lines as an alternative to animal models. For instance, the osmotic induction of the pathway for synthesis of the compatible organic osmolyte myo-inositol, which is evident in many O. mossambicus tissues, is fully reproducible in the OmB cell line derived from the brain of the tilapia O. mossambicus (38,(41)(42)(43).…”

Section: Significancementioning

confidence: 99%

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Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

Wang

Kültz

2017

Proc. Natl. Acad. Sci. U.S.A.

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Fish respond to salinity stress by transcriptional induction of many genes, but the mechanism of their osmotic regulation is unknown. We developed a reporter assay using cells derived from the brain of the tilapia Oreochromis mossambicus (OmB cells) to identify osmolality/salinity-responsive enhancers (OSREs) in the genes of O. mossambicus. Genomic DNA comprising the regulatory regions of two strongly salinity-induced genes, inositol monophosphatase 1 (IMPA1.1) and myo-inositol phosphate synthase (MIPS), was isolated and analyzed with dual luciferase enhancer trap reporter assays. We identified five sequences (two in IMPA1.1 and three in MIPS) that share a common consensus element (DDKGGAAWWDWWYDNRB), which we named "OSRE1." Additional OSREs that were less effective in conferring salinity-induced trans-activation and do not match the OSRE1 consensus also were identified in both MIPS and IMPA1.1. Although OSRE1 shares homology with the mammalian osmotic-response element/tonicity-responsive enhancer (ORE/TonE) enhancer, the latter is insufficient to confer osmotic induction in fish. Like other enhancers, OSRE1 trans-activates genes independent of orientation. We conclude that OSRE1 is a cis-regulatory element (CRE) that enhances the hyperosmotic induction of osmoregulated genes in fish. Our study also shows that tailored reporter assays developed for OmB cells facilitate the identification of CREs in fish genomes. Knowledge of the OSRE1 motif allows affinity-purification of the corresponding transcription factor and computational approaches for enhancer screening of fish genomes. Moreover, our study enables targeted inactivation of OSRE1 enhancers, a method superior to gene knockout for functional characterization because it confines impairment of gene function to a specific context (salinity stress) and eliminates pitfalls of constitutive gene knockouts (embryonic lethality, developmental compensation). biochemical evolution | compatible osmolytes | osmotic stress signaling | enhancer | CRE

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

Wang

Kültz

2017

Proc. Natl. Acad. Sci. U.S.A.

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Using ovarian and brain cell culture from the Mullet, Liza klunzingeri, to assess the inhibitory effects of benzo[a]pyrene on aromatase activity

Sheibani

Salamat

Movahedinia

et al. 2020

J of Applied Toxicology

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We assessed the toxic effects of benzo[a]pyrene (BaP) on cell viability, aromatase (Aro) activity and steroid production using ovarian and brain cell cultures obtained from Mullet, Liza klunzingeri. The brain and ovary were minced and digested, and the cells were suspended in Leibovitz's L-15 medium supplemented with 15% and 20% fetal bovine serum. The cell suspensions were seeded on 25-cm 2 cell-culture flasks at 1 × 10 6 cells/mL and incubated at 25 C for 2 weeks. A BaP concentration of 10 −5 mol/L was accepted as the half-maximal inhibitory concentration. Ovarian and brain cells were exposed to different concentrations of BaP [0 (control), 10 −6 , 2 × 10 −6 , 3 × 10 −6 mol/L] and incubated at 30 C. At different sampling times (0, 12, 24 and 48 h) 40 ng/10 5 cells of 1,4,6-androstatriene-3,17-dione (ATD) was added to each well. Aro activity, 17β-estradiol (E2) and ATD production were determined. The sensitivity of the cultivated ovarian and brain cells to BaP increased dose dependently. BaP was a potent inhibitor of Aro activity at 2 × 10 −6 and 3 × 10 −6 mol/L, both in the cultivated brain and ovarian cells at different sampling times, with 10 −6 mol/L BaP found to be the least potent Aro inhibitor. E2 production decreased from cultivated ovarian and brain cells treated by different concentrations of BaP. In conclusion, BaP is able to change the activity of Aro and disrupt the biosynthesis of estrogens, and thus affects reproduction in fish. K E Y W O R D Saromatase, benzo (a) pyrene, brain cell culture, Liza klunzingeri, ovarian cell culture, steroid

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Osmotic regulation and tissue localization of the myo‐inositol biosynthesis pathway in tilapia (Oreochromis mossambicus) larvae

et al. 2014

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The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol, which protects cells from salinity stress. We exposed tilapia larvae just after yolk sac resorption to various hypersaline environments and recorded robust induction of the enzymes that constitute the MIB pathway, myo-inositol-phosphate synthase (MIPS), and inositol monophosphatase 1 (IMPA1). Strong up-regulation of these enzymes is evident at both mRNA (quantitative real-time PCR) and protein (densitometric analysis of Western blots) levels. The highest level of induction of these enzymes occurs at the highest salinity that larvae were exposed to (90 ppt). Less severe salinity stress causes a proportionately reduced induction of the MIB pathway. Two distinct MIPS mRNA variants are present in tilapia larvae and both are induced at comparable levels for all the salinity challenges tested (34, 70, and 90 ppt). Immunohistochemical localization of IMPA1 protein in sagittal sections of salinity stressed and control larvae identified tissues that are particularly potent in inducing the MIB pathway. These tissues include the skin (epidermis), gills, eye (ciliary epithelium) and heart. In particular, the epidermis directly facing the external milieu showed a very strong induction of IMPA1 immunoreactivity. IMPA1 induction in response to salinity stress was not observed in other tissues suggesting that tilapia larvae may also utilize compatible organic osmolytes other than solely myo-inositol for osmoprotection. We conclude that the MIB pathway plays an important role in protecting multiple (but not all) tissues of tilapia larvae from hyperosmotic salinity stress.

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Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

Cited by 44 publications

References 84 publications

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

Using ovarian and brain cell culture from the Mullet, Liza klunzingeri, to assess the inhibitory effects of benzo[a]pyrene on aromatase activity

Osmotic regulation and tissue localization of the myo‐inositol biosynthesis pathway in tilapia (Oreochromis mossambicus) larvae

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