A new epithelial-like cell line from eye muscle of catla Catla catla (Hamilton): development and...

A new epithelial-like cell line from eye muscle of catlaCatla catla (Hamilton): development and

characterization

V. P. ISHAQ AHMED*, V. CHANDRA†, V. PARAMESWARAN*,C. VENKATESAN*, R. SHUKLA†, R. BHONDE†

AND A. S. SAHUL HAMEED*‡

*Aquaculture Division, Department of Zoology, C. Abdul Hakeem College,Melvisharam 632 509, Tamil Nadu, India and †Laboratory No. IV, National Centre for

Cell Science, Pune University Campus, Ganeshkhind, Pune, India

(Received 21 November 2007, Accepted 6 February 2008)

A new cell line [Sahul India Catla Eye (SICE)] has been developed from eye tissue of Indian major

carp (Catla catla), a freshwater fish cultivated in India. The cell linewasmaintained inLeibovitz’s L-

15 supplementedwith 15% foetal bovine serum (FBS). These cells have been subcultured>80 times

over a period of 1�5 years. This cell line has been designated SICE. The SICE cell line consists

predominantly of epithelial-like cells. These cells are strongly positive for epithelial markers such as

pancytokeratinandcytokeratin19.The cellswereable togrowat temperaturesbetween25and32°Cwith optimum temperature of 28° C. The growth rate of catla eye cells increased as the FBS

proportion increased from 2 to 20% at 28° C with optimum growth at the concentrations of 15 or

20%FBS.Sixmarinefish viruses (fishnervous necrosis virus,marinebirnavirus-NC1, chumsalmon

virus, infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus-Sp and hirame

rhabdovirus) were tested on this cell line to determine its susceptibility. After confluency, the cells

were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached

confluency on the fourth day after subculture. Polymerase chain reaction amplification of

mitochondrial 12S rRNA indicated identity of these cell lines with those reported from this animal

species, confirming that the cell lines were of catla origin. The cells were successfully cryopreserved

and revived at passage numbers 10, 25, 40 and 60. The cell cycle analysis by fluorescence-activated

cell sorter revealed that most of the cells on the second day of culture were in S-phase, indicating

a high growth rate. When the SICE cells were transfected with pEGFP vector DNA, significant

fluorescent signalswere observed suggesting that the SICEcell line canbeauseful tool for transgenic

and genetic manipulation studies. # 2008 The Authors

Journal compilation # 2008 The Fisheries Society of the British Isles

Key words: Catla catla; cell line; cryopreserved; eye; transfected; virus.

INTRODUCTION

The Indian major carps (catla, rohu and mrigal) are commercially importantfish species widely cultured in the entire Indian sub-continent. The growingcarp culture industry has suffered several incidences of mass mortality of carps

‡Author to whom correspondence should be addressed. Tel.: þ91 04172269487; email: cah_sahul@

hotmail.com

Journal of Fish Biology (2008) 72, 2026–2038

doi:10.1111/j.1095-8649.2008.01851.x, available online at http://www.blackwell-synergy.com

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Journal compilation # 2008 The Fisheries Society of the British Isles

in culture systems suspected to be caused by microbial diseases, particularly ofviral aetiology (Mohan & Shankar, 1994).Cell lines provide an important tool for carrying out virological, toxicologi-

cal, carcinogenic and cellular physiology and also gene regulation and expres-sion studies. To date, >150 fish cell lines have been developed for virusisolation and propagation (Fryer & Lannon, 1994). A large number of cell lineshave been established in freshwater fishes (Fryer & Lannon, 1994; Hong et al.,2004), but relatively few cell lines have been developed in India. Recently,continuous cell lines from gill tissue of mrigal Cirrhinus mrigala (Hamilton)(Sathe et al., 1995) and rohu Labeo rohita (Hamilton) (Sathe et al., 1997),kidney of barramundi Lates calcarifer (Bloch) (Sahul Hameed et al., 2006),spleen of barramundi (Parameswaran et al., 2006a) and embryos of barramundi(Parameswaran et al., 2006b) have been established. Primary cultures froma variety of organs such as heart tissue of Indian major carp (Rao et al.,1997), kidney of stinging catfish Heteropneustes fossilis (Burchell) (Singhet al., 1995), caudal fin of Tor putitora (Prasanna et al., 2000), caudal fin of rohu(Lakra & Bhonde, 1996), ovary of Clarias gariepinus (Hamilton) (Kumar et al.,2001) and fry of golden masheer (T. putitora) (Lakra et al., 2006) have also beenreported.Catla is a potential candidate species for farming in India and is in high

demand in the domestic market. It is extremely important to establish a contin-uous cell line for monitoring the viral and rickettsial diseases of fishes and forvaccine production against bacterial and viral pathogens. In India, detailedstudies on viral infections have not been carried out owing to lack of species-specific cell lines. Thus, a cell line in catla is urgently required for isolating andidentifying viruses that cause diseases in this species.In this study, a new cell line derived from the eye muscle of the Catla catla

(Hamilton) was established and characterized and the susceptibility of this cellline to some marine fish viruses was investigated. Some characteristics werealso described of cultured Sahul India Catla Eye (SICE) cells by immunocon-focal microscopy and fluorescence-activated cell sorter (FACS) analysis. Theefficiency of transfection and gene expression was also investigated in thisnew cell line.

MATERIALS AND METHODS

INITIATION OF PRIMARY CELL CULTURE ANDROUTINE MAINTENANCE

Normal and apparently healthy juveniles of catla C. catla (5–10 g in mass) werecollected from grow-out ponds of Induced Carps Spawning Centre (ICSC), Sathanur,Tamil Nadu, and transported live to the laboratory. In the laboratory, the animals weremaintained in sterile, aerated fresh water containing 1000 IU ml�1 penicillin and 1000mg ml�1 streptomycin overnight at room temperature (25–28° C). The fish were anaes-thetized in iced water, dipped in 5% chlorex for 5 min and wiped with 70% ethanol andoperated in vivo under aseptic conditions. The eye, brain, heart, spleen, kidney, liver andfin tissues of the fish were taken aseptically and washed three times in antibioticmedium containing Leibovitz’s L-15 (GIBCO, Invitrogen Corp., Carlsbad, CA, U. S. A.),500 IU ml�1 penicillin and 500 mg ml�1 streptomycin. The tissues were minced into small

A NEW EPITHELIAL-LIKE CELL LINE FROM EYE MUSCLE OF CATLA CATLA 2027

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Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 2026–2038

pieces (c. 1 mm3 in size) in Leibovitz’s L-15 with 500 IU ml�1 penicillin, 500 mg ml�1

streptomycin and 2�5 mg ml�1 fungizone. The tissue fragments were inoculated into 25cm2 cell culture flasks, and 5 ml of growth medium was added in each flask. The mediumL-15 was supplemented with 20% foetal bovine serum (FBS), 100 IU ml�1 penicillin, 100mg ml�1 streptomycin and 2�5 mg ml�1 fungizone. The flasks were incubated at 28° C, andthe medium was replaced every 4 days.

When the cells formed a monolayer, the old medium was removed and the cell sheetswere washed with phosphate-buffered saline (PBS) and dispersed with 0�25% trypsinsolution [0�25% trypsin and 0�02% ethylenediaminetetraacetic acid (EDTA) in PBS].The cells were resuspended in 10 ml of growth medium and then distributed intotwo flasks. The concentration of FBS in the L-15 medium was reduced to 15% for pas-sages 20–70 and then to 10% for subsequent passages.

GROWTH STUDIES

The effect of temperature and FBS concentration on cell growth was studied with theSICE cell line at the 40th passage level. Approximately 105 SICE cells were seeded into25 cm2 cell culture flasks and incubated at 28° C for 24 h to allow for cell attachment.Then batches of flasks were incubated at selected temperatures of 20, 28, 32, 37 and 40° Cfor growth tests. Every other day, duplicate flasks at each temperature were washedwith PBS twice after which 0�2 ml of 0�25% trypsin solution was added to each flask.When the cells rounded up, the cell density was measured microscopically using a hae-mocytometer and the numbers were expressed as cells per mm2. The experiment wascarried out for 5 days.

The growth response to different concentrations of FBS (2, 5, 10, 15 and 20%) oncell growth was assessed in duplicate 25 cm2 flasks using the same procedure as men-tioned above at 28° C.

CRYOPRESERVATION

Forty-eight hour-old cultures of SICE cells at the various passage levels (25th, 50thand 70th) were harvested by centrifugation and suspended in complete culture mediumcontaining 10% FBS and 10% dimethyl sulphoxide at a density of 106 cells ml�1. Thecell suspensions were dispensed into 2 ml plastic ampoules and kept initially at �20° Cfor 4 h and then at �75° C overnight and finally transferred into liquid nitrogen (�196° C).The frozen cells were recovered from storage after 1 or 6 month post-storage by thaw-ing quickly with running water at 28° C. Following removal of the freezing medium bycentrifugation, the cells were suspended in complete medium L-15 with 10% FBS andtested for viability by haemocytometer counting after trypan blue staining. The viablecells were seeded into a 25 cm2 cell culture flask and observed.

VIRAL SUSCEPTIBILITY

Six marine fish viral pathogens, namely fish nervous necrosis virus, marine birnavirus-NC1, chum salmon virus (CSV), infectious haematopoietic necrosis virus (IHNV), in-fectious pancreatic necrosis virus-Sp (IPNV-SP) and hirame rhabdovirus (HIRRV) wereselected for inoculation in SICE cell line at the 70th passage to observe the cytopathiceffect (CPE) microscopically. The preparation of these viruses for inoculation was car-ried out according to the procedure described by Kang et al. (2003) and Parameswaranet al. (2007). For infection, SICE cells from 25 cm2 flasks were inoculated in 24 wellplate to give a confluence of 60–70% and incubated for 12–24 h at 28° C. After re-moval of the medium, 0�1 or 1 ml of virus suspension at a dilution of 10�1 to 10�3

was inoculated into the cultured cells in a 24 well plate or flask and allowed to adsorbfor 55 min. Then 0�5 or 5 ml maintenance medium containing 10% FBS was added.The cells were incubated at 25° C and examined daily for the appearance of CPE for upto 2 weeks.

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IMMUNOFLUORESCENCE STAINING AND CONFOCALLASER-SCANNING MICROSCOPY

For immunophenotyping of the SICE cell line, the cells were grown on cover slipsfor 24 h, fixed with 3�7% p-formaldehyde for 10 min at 4° C, washed with PBS, per-meabilized with 0�1% Triton X-100 and blocked in PBS containing 1% bovine serumalbumin (BSA). Selected mouse and rabbit polyclonal antibodies, antihuman fibronectin,pancytokeratin, Ki67, cytokeratin 19 and desmin (Sigma, St Louis, MO, U.S.A.) werediluted 1:75 in PBS with 1% BSA and directly added to the fixed cells and kept for 2 hat room temperature (RT). Then the cells were washed with wash buffer, followed byaddition of the appropriate secondary antibody. Secondary antibodies, antimouse im-munoglobin G (IgG) fluorescein iso-thio cyanate (FITC) and antirabbit IgG FITC atthe dilution of 1:50 in the same solution as the primary antibody were applied for45 min at RT. Again the cells were washed with wash buffer and the cover slips weremounted with antifade 1, 4-diazobicyclo-2, 2, 2-octanex in mounting medium (Sigma).The cover slips were observed using a pinhole setting of 100 mm with confocal laser-scanning microscopy (CFLSM) (Carl Zeiss, Jena, Germany). Images were capturedby the CCD-4230 camera coupled with the microscope and processed using the com-puter-based programmable image analyzer KS300 (Carl Zeiss). Immunoconfocalmicroscopy of the cell cultures was performed as described by Anjali et al. (2003).

CELL-CYCLE ANALYSIS

FACS analysis was carried out for SICE cells at intervals of 12 h for 2 days witha plating cell count of 1 � 105 in each flask. The cells were trypsinized thereafter,washed twice with cold PBS and centrifuged. The cell pellets were resuspended in1 ml of ice-cold ethanol for 1 h at 4° C. The cells were centrifuged at 1200 g for 5min, pellet washed twice with cold PBS and resuspended in 100 ml RNase (5 mgml�1 final concentration) for 30 min at 37° C. Then 300 ml propidium iodide (PI) (stock50 mg ml�1 PI in PBS) was added and the mixture kept on ice for 1 h. Cells were ana-lysed on FACS Vantage (Becton-Dickson, Oakville, ON, Canada).

TRANSFECTION

SICE cells at 50th passages were propagated in a 6 well plate at a density of 5 � 105

cells well�1. Subconfluent monolayers were transfected with 2 mg of pEGFP eukaryoticexpression vector (Clontech, Carlsbad, CA, U.S.A.) using Lipofectamine 2000 (Invitrogen),and the green fluorescence signals were observed under a Leica fluorescence microscopeafter 24 h transfection (Qin et al., 2005).

RIBOSOMAL RNA GENE ANALYSIS BY POLYMERASECHAIN REACTION

Template DNA for polymerase chain reaction (PCR) assays was prepared by extrac-tion from tissues of SICE cells following the method described by Lo et al. (1996).Briefly, the samples were homogenized separately in sodium chloride-tris-EDTA (NTE)buffer [0�2 m NaCl, 0�02 m Tris–HCl and 0�02 m EDTA, pH 7�4] and centrifuged at3000 g at 4° C, after which the supernatant fluids were placed in fresh centrifuge tubestogether with an appropriate amount of digestion buffer (100 mm NaCl, 10 mmTris–HCl, pH 8�0, 50 mm EDTA, pH 8�0, 0�5% sodium dodecyl sulphate, 0.1 mg ml�1

proteinase K). After incubation at 65° C for 2 h, the digests were deproteinized by successivephenol/chloroform/iso-amyl alcohol extraction and DNA was recovered by ethanol pre-cipitation, drying and resuspension in Tris-EDTA (TE) buffer. The primers forward(59-GCCCATTTCTTTCCACCT-39) and reverse (59-CCACTATGCTTAGCCGTA-39)were used for amplifying 12S rRNA of C. catla. A fragment of 284 bp was amplified.


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PCR was conducted in an Eppendorf thermal cycler (Eppendorf, Wesseling, Germany).Each PCR reaction was conducted in a 25 m1 volume containing both forward andreverse primers (10 mM, 0�5 m1 each), MgCl2 (25 mM, 1�5 m1), dNTPs (2 mM, 2�0 m1),PCR buffer (10 � 2�5 m1), Taq-DNA (1 U), template DNA (0�3–0�4 m) and nucleic acidfree water. PCR cycling condition included an initial denaturation at 95° C for 5 min,followed by 35 cycles at 95° C for 30 s, annealing temperature at 55° C for 1 min,72° C for 30 s and a final extension of 10 min at 72° C. Amplified products were analysedin 1�2% agarose gel containing ethidium bromide and visualized with a ultraviolet trans-illuminator.

CHROMOSOME ANALYSIS

A SICE cell at passage 35 was used for chromosome analysis. The cells were inoc-ulated in a 25 cm2 culture flask and incubated for 24–36 h. Colchicine (0�04%)(Sigma) was added to the cells and incubated for 2 h in culture flasks. Cells wereremoved from the flask surface and centrifuged at 500 g for 5 min. The pellet wasgently resuspended in 0�027 M KCl and incubated at 25° C for 30 min. Cells were thencentrifuged again at 500 g for 5 min. Supernatants were discarded and cells resus-pended. Freshly mixed, cold 3:1 methanol–acetic acid fixative was added slowly whileaspirating the cell suspension gently. These fixed cells were then washed three timeswith fresh fixative and then resuspended in a small amount of fixative. The suspensionwas dropped onto glass slides, air-dried and stained with 5% Giemsa (pH 6�8) for 15–20 min. Chromosome counts were performed in >100 metaphase plates from bothpassages.

RESULTS

MORPHOLOGY OF CULTURED SICE CELLS

Cell cultures were initiated from several tissues of catla (C. catla), whichincluded eye, liver, spleen, kidney and gill tissues. The cells migrated fromthe different tissue fragments, grew well and formed monolayers during the firstmonth. However, only the cells from the eye muscle tissue grew continuouslywhen subcultured at intervals of 5–7 days. In primary culture, cells adheredwell and achieved confluence in 6 days at 28° C. Cells were subcultured inL-15 medium with 20% FBS at a ratio of 1:2 every 9 days for the initial10 passages. For the first 10 passages, 50% culture medium was replaced withfresh medium at 4 day intervals. After 10 subcultures, cells were subcultured ata ratio of 1:2 to 1:4 at 5–7 days interval, and FBS was reduced to 15% in theL-15 culture medium. To date, cells have been subcultured >80 times. The cellswere split at a ratio of 1:2 or 1:3. Morphologically, initial 10 subcultures of cellline consisted of both epithelial and fibroblast-like cells [Fig. 1(a)]. After 25subcultures, only epithelial-like cells were observed [Fig. 1(b)]. The cells weresplit at a ratio of 1:2 every 5–7 days after 20 subcultures. The eye cell linehas been subcultured >80 times since initiation in January 2006 and is desig-nated as SICE cell line. Morphologically, SICE cell line is composed of epithe-lial-like cells with diameters of 20–25 mm [Fig. 1(b)]. The majority of cells werefairly homogeneous in size, having a small chromatin pattern. The cells reachedconfluency on the fourth day of culture. Very few apoptotic cells were foundduring the microscopic observation of cells for apoptotic bodies.


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OPTIMAL GROWTH CONDITION

SICE cells exhibited different growth rates at different incubation tempera-tures between 24 and 32° C. However, maximum growth was obtained at28° C [Fig. 2(a)]. No significant growth was observed at 20, 37 and 40° C inthe cells. The growth rate of SICE cells increased as the FBS proportionincreased from 2 to 20% at 28° C. Cells exhibited poor growth at 2 and 5%concentrations of FBS, relatively good growth at 10% but maximum growthoccurred with the concentrations of 15 and 20% FBS [Fig. 2(b)].

CRYOPRESERVATION AND REVIVAL

The SICE cells were cryopreserved at 25th, 50th and 70th passages. The cellswere recovered from storage and grew to confluency within 5 days. The average

FIG. 1. Phase-contrast photomicrographs of the catla eye cell line [Sahul India Catla Eye (SICE)] at

(a) passage 10 (b) passage 70 (�200).


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viability of the cells after cryopreservation was estimated to be c. 70–80% withthe same morphology.

VIRAL SUSCEPTIBILITY OF SICE CELLS

The SICE cells were highly resistant to six marine fish viruses namely noda-virus, MABV, IPNV, IHNV, HIRRV and CSV. No significant symptoms norCPE were observed in the cells up to 2 weeks of observation when comparedwith control cells (data not shown).

IMMUNOCONFOCAL MICROSCOPICAL OBSERVATION

SICE cells were strongly positive for pancytokeratin, cytokeratin 19 and pro-liferative marker Ki67 [Fig. 3(a–d)].

FIG. 2. Growth response of the SICE cell line at the 40th passage to (a) selected temperature and (b) foetal

bovine serum (FBS) concentrations.


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CELL-CYCLE ANALYSIS AND TRANSFECTION EFFICIENCY

The DNA content of SICE cells in the S-phase cells (G2–M) observed on thesecond day of the subculture was determined (Fig. 4). When SICE cell lineswere successfully transfected with pCMV-EGFP by means of Lipofectamine2000, the expression of EGFP in SICE cell lines could be detected as earlyas 12 h after transfection (Fig. 5).

MITOCHONDRIAL-GENE ANALYSIS

To verify the origin of SICE cell line by heteroduplex analysis, DNA wasisolated from the SICE cells at their 25th passage. Amplification of the ex-tracted DNA using the primers designed from genomic sequence of 12S rRNAof C. catla revealed the expected PCR product of 284 bp (Fig. 6).

CHROMOSOME ANALYSIS

The results of chromosome counts of 100 metaphase plates from SICE cellline at passage 35 revealed that the chromosome numbers varied from 30 to 55.The modal number (2n) was 50 [Fig. 7(a), (b)].

FIG. 3. Confocal micrograph showing expression of SICE cell line (a) nucleus stain with 49, 6-diamidino-

2-phenylindole (DAPI blue), (b) pancytokeratin (red) nucleus stain with DAPI (blue), (c) Ki67 (red)

nucleus stain with DAPI (blue) and (d) cytokeratin 19 (green) nucleus stain with DAPI (blue).


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DISCUSSION

Successful production in the aquaculture industry has been increasingly ham-pered by many factors including diseases, especially those caused by viruses.Susceptible cell lines are essential for the isolation, cultivation and characteriza-tion of fish viruses. Since the first fish cell line reported in the literature in 1962(Wolf & Quimby, 1962), at least 157 fish cell lines have been established (Fryer &Lannon, 1994). Most of them were derived from freshwater or anadromous fishspecies. However, studies on fish cell lines are growing rapidly, reflecting theincreased interest in the cultivation of native species of Indian major carps.In India, only a few freshwater fish and marine fish cell lines and primary cell

FIG. 5. Expression of GFP gene in SICE cell lines at passage 50th transfected with pEGFP-N1. Bar:

20 mm.

FIG. 4. DNA content in second-day cultures of Sahul India Catla Eye (SICE) cell line at the 70th passage.

M1 ¼ G0–G1 (bigger peak) and M2 ¼ G2–M (smaller peak) population in each case, whereas the

M3 represents S-phase population of SICE cells.


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culture have been developed (Sathe et al., 1995; Lakra & Bhonde, 1996; Raoet al., 1997; Sahul Hameed et al., 2006). The present work describes the devel-opment and characterization of Indian major carp (C. catla), designatedas SICE, derived from eye of catla. This cell line is well adapted to grow in

FIG. 6. Agarose gel electrophoresis of PCR products from SICE cells. M, molecular mass marker; lane 1,

without template; lane 2, muscle tissue of catla; lane 3, SICE cell line.

FIG. 7. Chromosome number distribution at (a) passages 35 and (b) metaphase. In total, 100 metaphases

were counted.


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Leibovitz’s L-15 supplemented with FBS. The suitability of Leibovitz’s L-15 insupporting fish cell lines compared with that of other media has been docu-mented by Fernandez-Puentes et al. (1993a, b) when they compared the growthof many fish cell lines in different culture media at different temperatures andsodium chloride concentrations. The SICE cell line has been maintainedthrough >80 passages over a 1�5 year period.The growth temperature range for SICE was 20–32° C with optimum growth

at 28° C, which was identical with other fish cell lines reported previously(Nicholson et al., 1987; Tong et al., 1997, 1998; Kang et al., 2003; SahulHameed et al., 2006). One of the advantages of cell lines that grow over a widetemperature range is their potential suitability for isolating both warmwaterand coldwater fish viruses (Nicholson et al., 1987). The growth rate of SICEcells increased as the FBS concentration increased from 2 to 20%. However,a 10% concentration of FBS also provided relatively good growth, and thisis an advantage to maintain this cell line at low cost (Ye et al., 2006). Cryopres-ervation of cell lines is necessary for long-term storage. The feasibility of cryo-preservation of this cell line was demonstrated, with appreciable recovery afterthawing of up to 70–80%, while that of SAF-1 [gilthead sea bream (Sparusaurata L.), fin–], GF-1 and SF was 50% (Bejar et al., 1997), 73% (Chi et al.,1999) and 80–85% (Chang et al., 2001), respectively.Antibodies of epithelial, proliferative, cytokeratin 19 and Ki67 and markers

were used to confirm the epithelial and proliferative nature of this cell line.SICE cells showed strong positive to pancytokeartin, cytokeratin 19 and pro-liferative Ki67 markers. These results show that SICE cell line is of epithelialtype and highly proliferative.Susceptibility of cell lines to viral infection is the basis for isolating and char-

acterizing fish viruses. In the present study, six marine fish viruses were testedon SICE cell line to determine its resistance to these viruses and the resultsshowed that this cell line was not susceptible to any of the six tested marinefish viruses. This has been confirmed by observation of CPE. Previous attemptsto isolate nodavirus in a variety of cell lines were largely unsuccessful (Breuilet al., 1991; Mori et al., 1991; Munday et al., 1992).The FACS analysis showed that a higher percentage of cells were found to

be in the S-phase on the second day of culture. The DNA content of the cellline revealed a diploid cell population, which is of great interest for cytogeneticstudies.Mitochondrial DNA has frequently been documented in literature for species

identification, classification and phylogenetic relationships among aquatic andmammalian species. The 12S rRNA has been particularly used. To further con-firm the origin of the newly established SICE, cellular DNA was extractedfrom these cells at early passage 25th and amplified by PCR using specificprimer pairs targeting the conserved regions of 12S rRNA of catla. Karyotypeanalysis revealed that the SICE cell line possessed a modal chromosome num-ber of 2n ¼ 50, which was identical with the reported modal chromosome num-ber of this species (Majumdar & Ray-Chaudhuri, 1976).In conclusion, a healthy cell line (SICE) was developed from eye muscle of

catla. The SICE cell line is highly resistant to non-specific marine fish virusesand can be used as a diagnostic tool for isolation, propagation and detection


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of viruses specific to catla. The SICE cells will also facilitate the developmentcell models for toxicological studies to replace whole animals and genetic engi-neering.

The authors thank the management of C. Abdul Hakeem College for providing thefacilities to carry out this work. The authors also thank the official persons of InducedCarps Spawning Center (ICSC), Sathanur, Tamil Nadu, for providing the healthyexperimental animals. This work was funded by Department of Biotechnology, Govern-ment of India, New Delhi, India.

References

Anjali, S., Bhosale, A., Shepal, V., Ravi, S., Baburao, V. S., Prabhakara, K. & Padma, S.(2003). A unique model system for tumor progression in GBM comprising twodeveloped human neuro-epithelial cell lines with differential transforming potentialand coexpressing neuronal and glial markers. Neoplasia 5, 520–532.

Bejar, J., Borrego, J. J. & Alvarez, M. C. (1997). A continuous cell line from the culturedmarine fish gilt-head seabream (Sparus aurata). Aquaculture 150, 143–153.

Breuil, G., Bonami, J. R., Pepin, J. F. & Pichor, Y. (1991). Viral infection (picorna-likevirus) associated with mass 574 mortalities in hatchery-reared seabass (Dicen-trarchus labrax) larvae and juveniles. Aquaculture 97, 109–116.

Chang, S. F., Ngoh, G. H., Kuch, L. F. S., Qin, Q. W., Chen, C. L., Lam, T. J. & Sin, Y. M.(2001). Developmental of a tropical marine fish cell line from Asian seabass (Latescalcarifer) for virus isolation. Aquaculture 192, 133–145.

Chi, S. C., Hu, W.W. & Lo, B. J. (1999). Establishment and characterization of a continuouscell line (GF-1) derived from grouper, Epinephelus coioides: a cell line susceptible togrouper nervous necrosis virus (GNNV). Journal of Fish Diseases 22, 173–182.

Fernandez-Puentes, C., Novoa, B. & Figueras, A. (1993a). Initiation of a cell line fromturbot (Scophthalmus maximus L.). In Vitro Cellular & Developmental Biology –Animal 29, 899–900.

Fernandez-Puentes, C., Novoa, B. & Figueras, A. (1993b). A new fish cell line derivedfrom turbot (Scophthalmus maximus L.) TV-1. Bulletin of the European Associationof Fish Pathologists 13, 94–96.

Fryer, J. L. & Lannon, C. N. (1994). Three decades of fish cell culture: a current listing ofcell lines derived from fish. Journal of Tissue Culture Methods 16, 87–94.

Hong, Y., Chen, S. L., Gui, J. F. & Schartl, M. (2004). Retention of the developmentalpluripotency in medaka embryonic stem cells after gene transfer and long-termdrug selection towards for gene targeting in fish. Transgenic Research 13, 41–50.

Kang, M. S., Oh, M. J., Kim, Y. J., Kawai, K. & Jung, S. J. (2003). Establishment andcharacterization of two cell lines derived from flounder, Paralichthys olivaceus(Temminck & Schlegel). Journal of Fish Diseases 26, 657–665.

Kumar, G. S., Singh, I. B. S. & Philip, R. (2001). Development of a cell culture system fromthe ovarian tissue of African catfish (Clarias gariepinus). Aquaculture 194, 51–62.

Lakra, W. S. & Bhonde, R. R. (1996). Development of primary cell culture from thecaudal fin of an Indian major carp, Labeo rohita (Ham.). Asian Fisheries Science 9,149–152.

Lakra, W. S., Bhonde, R. R., Sivakumar, N. & Ayyappan, S. (2006). A new fibroblastlike cell line from the fry of golden mahseer Tor putitora (Ham). Aquaculture 253,238–243.

Lo, C. F., Leu, J. H., Ho, C. H., Chen, C. H., Peng, S. E., Chen, Y. T., Yeh, P. Y.,Huang, C. J., Wang, C. H. & Kou, G. H. (1996). Detection of baculovirusassociated with white spot syndrome (WSSV) in penaeid shrimps using polymerasechain reaction. Diseases of Aquatic Originisms 25, 133–141.

Mohan, C. V. & Shankar, K. M. (1994). Epidemiological analysis of epizootic ulcerativesyndrome of fresh and brackishwater fishes of Karnataka, India. Current Science66, 656–658.


# 2008 The Authors


Mori, K., Nakai, T., Naghahara, M., Muroga, K., Mckuchi, T. & Kanno, T. (1991). Aviral disease in hatchery-reared larvae and juveniles of redspotted grouper. FishPathology 26, 209–210.

Munday, B. L., Langdon, J. S., Hyatt, A. & Humphrey, J. D. (1992). Mass mortalityassociated with a viral-induced vacuolating encephalopathy and retinopathy oflarval and juvenile barramundi, Lates calcarifer (Bloch). Aquaculture 103, 197–211.

Nicholson, B. L., Danner, D. J. & Wu, J. L. (1987). Three new continuous cell lines frommarine fishes of Asia. In Vitro Cellular & Developmental Biology 23, 199–204.

Parameswaran, V., Shukla, R., Bhonde, R. R. & Sahul Hameed, A. S. (2006a). Spleniccell line from sea bass, Lates calcarifer: establishment and characterization.Aquaculture 261, 43–53.

Parameswaran, V., Shukla, R., Bhonde, R. R. & Sahul Hameed, A. S. (2006b).Establishment of embryonic cell line from sea bass (Lates calcarifer) for virusisolation. Journal of Virological Methods 137, 309–316.

Parameswaran, V., Ishaq Ahmed, V. P., Shukla, R., Bhonde, R. R. & Sahul Hameed,A. S. (2007). Development and characterization of two new cell lines from milkfish(Chanos chanos) and grouper (Epinephelus coioides) for virus isolation. MarineBiotechnology 9, 281–291.

Prasanna, I., Lakra, W. S., Ogale, S. N. & Bhonde, R. R. (2000). Cell culture from finexplant of endangered mahseer Tor putitora (Hamilton). Current Science 79, 93–95.

Qin, Q. W., Wu, T. H., Jia, T. L., Hegde, A. & Zhang, R. Q. (2005). Development andcharacterization of a new tropical marine fish cell line from grouper, Epinepheluscoioides susceptible to iridovirus and nodavirus. Journal of Virological Methods131, 58–64.

Rao, K. S., Joseph, M. A., Shanker, K. M. & Mohan, C. V. (1997). Primary cell culturefrom explants of heart tissue of Indian major carps. Current Science 73, 374–375.

Sahul Hameed, A. S., Parameswaran, V., Shukla, R., Singh, B. & Bhonde, R. R. (2006).Establishment and characterization of India’s first marine fish cell line from kidneyof sea bass, Lates calcarifer. Aquaculture 257, 92–103.

Sathe, P. S., Maurya, D. T., Basu, A., Gogate, S. S. & Banerjee, K. (1995). Establish-ment and characterization of a new fish cell line, MG-3, from the gills of mrigalCirrhinus mrigala. Indian Journal of Experimental Biology 33, 589–594.

Sathe, P. S., Basu, A., Mourya, D. T., Marathe, B. A., Gogate, S. S. & Banerjee, K.(1997). A cell line from the gill tissues of Indian cyprinoid Labeo rohita. In VitroCellular & Developmental Biology – Animal 33, 425–427.

Singh, I. S. B., Rosamma, P., Raveendranath, M. & Shanmugam, J. (1995). Developmentof primary cell cultures from kidney of freshwater fish Heteropneustus fossilis.Indian Journal of Experimental Biology 33, 595–599.

Tong, S. L., Lee, H. &Miao, H. Z. (1997). The establishment and partial characterizationof a continuous fish cell line FG-9307 from the gill of flounder Paralichthysolivaceus. Aquaculture 156, 327–333.

Tong, S. L., Miao, H. Z. & Li, H. (1998). Three new continuous fish cell lines of SPH,SPS and RSBF derived from sea perch (Lateolabrax japaonicus) and red sea bream(Pagrosomus major). Aquaculture 169, 143–151.

Wolf, K. & Quimby, M. C. (1962). Established eurythermic line of fish cells in vitro.Science 135, 1065–1066.

Ye, H. Q., Chen, S. L., Sha, Z. Q. & Xu, M. Y. (2006). Development and characterizationof cell lines from heart, liver, spleen and head kidney of sea perch (Lateolabraxjaponicus). Journal of Fish Biology 69, 115–126.

Electronic Reference

Majumdar, K. C. & Ray-Chaudhuri, S. P. (1976). Studies on the chromosomes of Indianmajor carps. In Proceedings of the Symposium Modern Trends in Zoological Researchin India, Calcutta, 26–27 July, pp. 68–69. http://www.fishbase.org/References/FBRefSummary.php?id=34798&speccode=4494


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