• Fisheries and Aquatic Sciences
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• 제16권3호
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• pp.177-185
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• 2013

The development of species-specific fish cell lines has become a valuable tool for biological research. In recent years, marine medaka Oryzias dancena has been recognized as a good experimental model fish but there are no reports of establishment of cell lines from this fish. In this study, two cell lines from O. dancena blastula embryos were established from 41 total trials (4.9%). The two cell lines displayed typical in vitro morphology and have been cultured for >121 passages, which corresponds to 293 days. The doubling times of the cell lines were 29.84 and 28.59 h, respectively, and both possessed the potential to expand in a clonal manner, albeit with significant differences between the two cell lines. The absence of any of the four main medium supplements; i.e., fish serum, fetal bovine serum, basic fibroblast growth factor, and medaka embryo extract, significantly inhibited growth. The proportion of cells possessing normal chromosome number was 45% and 46.7% of the cell lines, respectively. Taken together, two cell lines that proliferate continuously were established from marine medaka and these cell lines may provide a basic tool for characterizing the unique features of this fish species.

• 한국동물생명공학회지
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• 제35권1호
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• pp.65-72
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• 2020

Fish ovarian germline stem cells (OGSCs) that have the abilities to self-renew and differentiate into functional gametes can be used in various researches and applications. A main issue to be solved for effective utilization of fish OGSCs is the development of their stable in vitro culture condition, but only few researches about fish OGSC culture have been reported so far. In this study, in order to find the clues to develop the culture condition for OGSCs from Japanese medaka (Oryzias latipes), we tried to establish somatic cell lines as a candidate for the feeder cells and evaluated its supporting effects on the culture of ovarian cell populations from O. latipes. As the results, the somatic cell lines could be established only from the embryonic tissues among three tissues derived from embryos, fins and ovaries. Three embryonic cell lines were tested as a feeder cell for the culture of ovarian cell population and all three cell lines induced cell aggregation formation of the cultured ovarian cells whereas the feeder-free condition did not. Furthermore, a significant cellular proliferation was observed in the ovarian cells cultured on two of three cell lines. As a trial to increase the capacity of the cell lines as a feeder cell that supports the proliferation of the cultured ovarian cells, we subsequently established a stable line that expresses the foreign O. latipes fibroblast growth factor 2 (FGF2) from an embryonic cell line and evaluated its effectiveness as a feeder cell. The ovarian cells cultured on FGF2 expressing feeder cells still formed cell aggregates but did not show a significant increase in cellular proliferation compared to those cultured on non-transformed feeder cells. The results from this study will provide the fundamental information for in vitro culture of medaka OGSCs.

• Journal of Microbiology
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• 제34권3호
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• pp.263-263
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• 1996

Infection of fish cells with IHNV resulted in gradual increase in cytosolic free $Ca^{2+}$ concentration $([Ca^{2+}]_i)$ in CHSE, gradual decrease in $[Ca^{2+}]_i$ in FHM, and no significant change in RTG cells. The degree of $[Ca^{2+}]_i$ increase or decrease was dependent on the amount of infectious virus, and these $[Ca^{2+}]_i$ variations were maximal at 16 hours after virus infection (p. i.) in both cell lines. When the fish cells were infected with inactivated IHNV, evident variation in $[Ca^{2+}]_i$ was not observed. Thus, infectivity of IHNV appears to correlate with changes in $[Ca^{2+}]_i$ in virus-infected cells. These IHNV-induced $[Ca^{2+}]_i$ changes were partially blocked by cycloheximide, but not affected by cordycepin. It seems to be that virus-induced $Ca^{2+}$ variations were more related with protein synthesis than RNA synthesis. Various $Ca^{2+}$ related drugs were used in search for the mechanisms of the $[Ca^{2+}]_i$, changes following IHNV infection of CHSE cells. Decreasing extracellular $Ca^{2+}$ concentration or blocking $Ca^{2+}$ influx from extracellular media inhibited the IHNV-induced increase in $[Ca^{2+}]_i$, in CHSE cells. Similar results were obtained with intracellular $Ca^{2+}$ blockers. Thus it is suggested that both the extracellular and the intracellular $Ca^{2+}$ sources are important in IHNV-induced $[Ca^{2+}]_i$ increase in CHSE cells.

• Journal of Microbiology
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• 제34권3호
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• pp.253-269
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• 1996

Infection of fish cells with IHNV resulted in gradual increase in cytosolic free Ca$\^$2+/ concentration ([Ca$\^$2+/)] in CHSE, gradual decrease in [Ca$\^$2+/] in FHM, and no significant change in RTG cells. The degree of [Ca$\^$2+/] increase or decrease was dependent on the amount of infectious virus, and these [Ca$\^$2+/] variations were maximal at 16 hours after virus infection (p. i.) in both cell lines. When the fish cells were infected with inactivated IHNV, evident variation in [Ca$\^$2+/] was not observed. Thus, infectivity of IHNV appears to correlate with changes in [Ca$\^$2+/] in virus-infected cells. These IHNV-induced [Ca$\^$2+/] changes were partially blocked by cycloheximide, but not affected by cordycepin. It seems to be that virus-induced Ca$\^$2+/ variations were more related with protein synthesis than RNA synthesis. Various Ca$\^$2+/ related drugs were used in search for the mechanisms of the [Ca$\^$2+/], changes following IHNV infection of CHSE cells. Decreasing extracellular Ca$\^$2+/ concentration or blocking Ca$\^$2+/ influx from extracellular media inhibited the IHNV-induced increase in [Ca$\^$2+/], in CHSE cells. Similar results were obtained with intracellular Ca$\^$2+/ sources are important in IHNV-induced [Ca$\^$2+/] increase in CHSE cells.

• 한국어병학회지
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• 제30권2호
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• pp.71-78
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• 2017

The glycoprotein of novirhabdoviruses is known to play a critical role in the determination of host specificity. Viral hemorrhagic septicemia viruses (VHSVs) in different genotypes have different glycoprotein sequences and show different preferences for specific cell lines. In this study, to know whether the glycoprotein is solely responsible for the host cell preference of VHSV, a recombinant VHSV expressing vesicular stomatitis virus (VSV) glycoprotein instead of VHSV IVa glycoprotein (rVHSV-VSV-G) was generated by reverse genetics and inoculated into several fish cell lines, then, cytopathic effect (CPE) and viral growth caused by rVHSV-VSV-G infection were compared with those caused by rVHSV-wild that was previously generated and has the same genomic sequence with wild-type VHSV except a few nucleotides. The plaque numbers of rVHSV-VSV-G were significantly higher in EPC, BF-2 and GF cells than those of rVHSV-wild. However, in HINAE cells (originated from olive flounder), rVHSV-VSV-G titer was significantly lower than rVHSV-wild titer, and both recombinant VHSVs were not grown well in CHSE-214 cells. Although statistical significances were detected in the titers between rVHSV-wild and rVHSV-VSV-G in several cell lines, the cell line-preference order of rVHSV-VSV-G was not different from that of rVHSV-wild. These results suggest that the replacement of VHSV glycoprotein may not completely change host cell preference, and other regions of VHSV might also involve in the determination of host cell preference.

• 한국어병학회지
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• 제11권2호
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• pp.89-96
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• 1998

해양버나바이러스(MABV)는 여러 종의 해양생물에 감염되며 숙주역이 넓다. 다양한 종의 숙주에 감염되었을 때의 MABV의 감염 특성을 규명하기 위하여, 주화세포 내에서 바이러스를 10대 계대 배양하여 in vitro로 연구했다. CHSE-214, RTG-2와 RSBK-2세포에서는 전형적인 CPE를 보이며 많은 양의 바이러스가 생산되었고, 높은 바이러스 단백질의 발현도 관찰되었다. 이에 반하여, EPC, FHM과 BF-2세포에서는 형광항체법에 의하여 바이러스단백질은 검출되었으나 CPE는 나타나지 않았다. EPC와 FHM 세포에서는 계대를 할수록 바이러스의 역가가 높아져, 바이러스의 숙주세포에의 적응이 일어난 것으로 보인다. 플라크의 크기는 CHSE-214, RTG-2와 RSBK-2세포에서 계대한 것이 다른 세포에서 계대한 것보다 커, 숙주세포의 종류에 따른 변이가 바이러스에 일어난 것으로 추측되었다. 게놈분절 A에 존재하는 VP2/NS 경계영역의 염기배열에서는 195번째의 염기에 특이적인 변이가 보였다. 숙주세포의 종류에 따라 다른 MABV의 감염특성은 자연계에서 다양한 숙주종에서 일어나는 in vivo에서의 감염특성을 반영하는 것으로 생각된다.

• 한국어류학회지
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• 제20권1호
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• pp.1-6
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• 2008

제브라피쉬 배아로부터 확보한 세포주로부터 외부형태와 세포 크기에 따라 3종류의 동형세포주를 확립하였다. 활발하게 증식하는 안정된 세포주 및 이들로부터 확립된 동형세포주의 세포특성은 변하지 않고 지속적으로 유지되었으며, 안정된 세포주로부터 총 18개의 콜로니를 확보하여 배양한 다음 3종류의 동형세포주를 선별하여 세포 특성을 분석하였다. 대부분의 동형세포주는 약 80% 정도 정상적인 염색체(2N=50)를 가지고 있었으며 FACs 분석과 일치하였다. 배아로부터 확립된 동형세포주에 항체테스트 결과, vimentin에서 양성을 보이는 결과로 볼 때 확립된 세포주는 분화된 섬유세포임이 확인되었다. 이러한 결과는, 확립된 동형세포주를 이용한 유전자조작과 어류복제에의 활용도를 높일 수 있음을 시사한다.

• 한국어병학회지
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• 제15권1호
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• pp.9-16
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• 2002

The objective of the study was to clarify the mechanism of persistent infection of marine birnavirus (MABV) in various nonpermissive cell lines. It was observed in CHSE-214, RTG-2 and RSBK-2 that the virus produced at high yield with typical cytopathic effect (CPE). On the contrary, the CPE was not produced in EPC, FHM and BF-2 cells. However amount of virus protein in both permissive and nonpermissive cell lines detected by ELISA was almost the same. Electron microscopy showed virions in permissive cells but not in nonpermissive cells. From the results, it is clear that virus protein and RNA were produced in nonpermissive cells as observed in permissive cells; however, assembly of the virus particles did not occur in nonpermissive cells.

• 한국어병학회지
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• 제6권2호
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• pp.205-218
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• 1993

On the development of hirame(Paratichtys olivaceus) culture, outbreak of scuticociliata infection was reported to cause severe damage in Japan. To establish effective measures for isolation and cultivation of this ciliate, we tried to culture this pathogenic ciliate using medium for bacteria and fish cell lines in vitro. Scuticociliata from the brain tissues of infected fish was aseptically inoculated to CHSE-214 cells cultured in MEM-10 without antibiotic. Scuticociliata grew well and the number of ciliate reached $10^6\;cells/ml$ at temperatures of $15^{\circ}C$ to $20^{\circ}C$ for 10d. The number of ciliate cultured in the cell lines is 10 times higher than the numbers cultured in the liquid medium alone. This ciliata could be cloned by dilution method. Scuticociliata isolated could grow well on 42 different cell lines that were established from marine fish, warm freshwater fish, and salmonids. This ciliate could be preserved in liquid nitrogen for more than 6 months. Subsequently, we observed the optimal temperature and salinity for growth, and tested the sensitivities of this organism to formaldehyde, flagyl(Metronidazole), Ekuteshin(Combination compound of sulfamonometoxin and ormethoprim), and ozonixation. Optimal temperature for growth was $25^{\circ}C$ and salinity was 1.0 to 1.5%. Washed scuticociliata was killed by formaldehyde at the concentration of 50ppm for 10min, but was not completely killed even at a high concentration of 400ppm for 20min in MEM-5. Flagyl and Ekuteshin can inhibit the growth of scuticociliata at the concentration of 1,000 and $100{\mu}g/ml$ in MEM-10, respectively. More than 99% of this scuticociliata could be killed by ozonization at a dose equivalent to $1.0mg/\ell$ oxidant for 30sec in sea water. Isolated scuticociliata showed the pathogenicity to the cultured hirame by artificial infection(I. P. injection, $10^5\;cells$/fish). The number of scuticociliata in the water could be counted by most probable number(MPN) method using tissue culture, and the minimum detectable number was $1.8\;cells/\ell$. The number in the reservoir tank for water supply to the culture tank was 110 cells/l. After cleaning by elimination of the sediments from of the reservoir tank and disinfected with formaldehyde, number of scuticociliata decreased and was counted less than $1.8\;cells/\ell$ and infection rate of cultured hirame was decreased.

• 한국해양생명과학회지
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• 제8권2호
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• pp.186-189
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• 2023

Uncoupling protein 1 (UCP1) is a unique mitochondrial membranous protein expressed in brown adipose tissue (BAT) in mammals. While its expression in response to cold temperatures and adipogenic inducers is well-characterized in mammals and human infants, the molecular characterization and expression of UCP1 in fish remain unexplored. To address this gap, we analyzed UCP1 expression in response to adipogenic inducers in a fish cell line, rainbow trout gonadal cells (RTG-2), and compared it with UCP1 expression in three mammalian preadipocytes, 3T3-L1, T37i, and WT1 exposed to the Peroxisome proliferator-activated receptor gamma (PPARγ) agonists, rosiglitazone (Rosi). In mammalian preadipocytes, UCP1 protein was highly expressed by Rosi, with an induction of adipogenesis observed in a time-dependent manner. This suggests that UCP1 plays a significant role in adipogenesis in mammals. However, RTG-2 cells showed no response to adipogenic inducers and exhibited only marginal expressions of UCP1. These results imply that RTG-2 cells may lack crucial responsive mechanisms to adipogenic signals or that the adipogenic response is regulated by other mechanisms. Further studies are needed to confirm these phenomena in fish preadipocytes when an appropriate cell line is established in future research.