Fish Physiology and Biochemistry

, Volume 43, Issue 6, pp 1477–1486 | Cite as

Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study

  • Ruoqing Wang
  • Nianwei Zhang
  • Renkai Wang
  • Shengpeng Wang
  • Na Wang
Article
  • 216 Downloads

Abstract

In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

Keywords

Japanese flounder (Paralichthys olivaceusAlbinism JFSK_wt JFSK_alb Nervous necrosis virus (NNV) 

Notes

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31472273), and the Taishan Scholar Climb Project of Shandong Province.

Authors’ contributions

NW conceived and designed the experiments. NW and RQW analyzed the experimental results and wrote the paper. NWZ conducted the primary cell culture. NW, RQW, and RKW subcultured JFSK_wt and JFSK_alb cell lines. RQW and NW conducted chromosome analysis, virus infection experiments, reporter gene and siRNA transfection assay, and qRT-PCR experiments. SPW provided the NNV infected grouper. All authors have read and approved the final manuscript.

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Ruoqing Wang
    • 1
    • 3
  • Nianwei Zhang
    • 1
    • 3
  • Renkai Wang
    • 1
    • 3
  • Shengpeng Wang
    • 1
  • Na Wang
    • 1
    • 2
  1. 1.Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of AgricultureYellow Sea Fisheries Research Institute, Chinese Academy of Fishery SciencesQingdaoChina
  2. 2.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and TechnologyQingdaoChina
  3. 3.College of Fisheries and Life ScienceShanghai Ocean UniversityShanghaiChina

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