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

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.

Betanodavirus and VER Disease: A 30-year Research Review

“…Other cell cultures derived from the brain of other fish species are: LJB, developed from sea perch (already mentioned), Chinese perch brain (CPB) cells from mandarin fish (Siniperca chuatsi), Trachinotus ovatus brain (TOGB) cells from golden pompano and DLB-1, also previously mentioned, from European sea bass [232,255,256]. Moreover, OLHNI cells derived from the caudal fin of medaka (Oryzias latipes), Trachinotus ovatus head kidney (TOHK) and trachinotus ovatus kidney TOK from golden pompano, Japanese flounder skin (JFSK), SISK and SISS from the kidney and spleen, respectively, of Asian sea bass/barramundi [257][258][259][260][261][262] have also been reported to be suitable for NNV propagation. It should be noted that all these susceptibility assays have been performed only with RGNNV strains, probably because this is the predominant genotype in Asian countries, where all these cell lines have been developed.…”

Characterization of the continuous skin fibroblastoid cell line, WE‐skin11f, from walleye (Sander vitreus)

“…This distinction is not always clear, as has been noted for fin cell lines as well (Mauger et al, ). In the fish skin invitrome, epithelioid cell lines have been described from lake sturgeon and mudloach (Vo et al, ; Xu, Sivaramasamy, Jin, Li, & Xiang, ) and fibroblastoid ones, from rainbow trout and Japanese flounder (Ossum, Hoffmann, Vijayan, Holt, & Bols, ; Wang, Zhang, Wang, Wang, & Wang, ). Other skin cell lines from goldfish (Lee, Caldwell, & Gibbons, ), Leopard wrasse (Ma, Sun, Zeng, & Lu, ), red‐spotted grouper (Lei et al, ) and Ussuri catfish (Ou, Lei, He, Zhou, & Zhang, ) have been described as showing both morphologies, with the proportions of cells in cultures having a particular shape changing in complex ways.…”

Development and characterization of a skin cell line from Chinese perch (Siniperca chuatsi) and its application in aquatic animal viruses

“…The two cell morphologies have been existed in previous established fish skin cell lines. For example, the skin cells derived from lake sturgeon (Vo et al, 2018), mud Loach (Xu et al, 2016) and Ussuri catfish (Ou et al, 2014) displayed epithelial‐like shape, while the skin cells from Japanese flounder (Wang et al, 2017), leopard wrasse (Ma et al, 2013) and rainbow trout (Kim et al, 2018) were fibroblast‐like. There was also fish skin cell line that had cell shape changes during subculture (Lei et al, 2012).…”

“…The cutaneous tissue of fish is an important physical and physiological barrier against invading pathogens. Although cell lines from skins of some fishes, such as red‐spotted grouper ( Epinephelus akaara ), Ussuri catfish ( Pseudobagrus ussuriensis ), Japanese flounder ( Paralichthys olivaceus ) and mosquitofish ( Gambusia affinis ), have been established (Jyotsna, 2020; Lei et al, 2012; Ou et al, 2014; Wang et al, 2017), the number of cell lines from fish skin remains relatively limited.…”