Modal Chromosome Number Research Articles

Development of a sea perch (Lateolabrax japonicus) fry cell line and its application to the study of fish virus-host interactions

We developed and characterized a new cell line from sea perch (Lateolabrax japonicus) fry (SPF). SPF cells were subcultured over 100 passages, and sequencing of the mitochondrial 18 s rRNA gene confirmed that SPF cells derived from Lateolabrax japonicus. SPF cells consisted predominantly of epithelial-like cells, and proliferated well in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome number was 48 at passage 55. Green fluorescence could be observed in pEGFP-N3 transfected SPF cells at passages 55. SPF cells showed susceptibility to sea perch iridovirus (SPIV), Singapore grouper iridovirus (SGIV), and red-spotted grouper nervous necrosis virus (RGNNV), which was demonstrated by the occurrence of severe cytopathic effects, increased viral protein synthesis, and abundant virus particles in the cytoplasm. Moreover, SPIV and SGIV infection in SPF cells caused profound alterations in the actin cytoskeleton, as evidenced by a significant decrease in the number of actin stress fibers. Of note, no apoptotic bodies were observed in either SGIV- or SPIV-infected cells, suggesting that these two viruses induced non-apoptotic cell death in vitro. Interferon-related genes were up-regulated in SPIV- or RGNNV-infected SPF cells, suggesting that the host interferon immune response was involved in SPIV or RGNNV infection. These results showed that our newly established SPF cell line was a useful tool for genetic manipulation, virus isolation, and functional studies of host-virus interactions.

Establishment and characterization of a continuous cell line from heart of Nile tilapia Oreochromis niloticus and its susceptibility to tilapia lake virus

In the present study, we have developed a continuous cell line from the heart tissue of the Oreochromis niloticus and used for studying susceptibility to tilapia lake virus (TiLV). The cell line, designated as OnH, has been subcultured up to 82 passages. The optimal growth of OnH cells was observed at 28–32 °C in iL-15 medium supplemented with 20 % fetal bovine serum. Karyotype analysis revealed that the modal chromosome number of OnH cells was 44. Partial amplification and sequencing of 16S rRNA gene confirmed the origin of OnH cell line from O. niloticus. Immunophenotyping revealed that OnH cells were of epithelial origin. These cells were successfully transfected with pAcGFP1-N1 mammalian expression vector. OnH cells showed cytopathic effects following inoculation with TiLV. The virus titration study indicated that the cells were highly susceptible to TiLV with TCID50 value of 105.3/mL. The qRT-PCR studies revealed that the optimal temperature for TiLV replication in OnH cells was 28 °C. Further, transmission electron microscopy of TiLV-infected OnH cells showed a number of electron-dense virus particles measuring 60−90 nm diameter, which were enclosed in the vesicles in the cytoplasm. Therefore, the newly established OnH cell line provides a valuable tool for isolation of viruses from disease cases suspected to be of viral etiology in this candidate species' and also for transgenic and genetic manipulation studies.

Establishment and characterization of a cell line derived from fin of the endangered Yangtze sturgeon (Acipenser dabryanus).

Yangtze sturgeon (Acipenser dabryanus) is an endangered endemic freshwater fish of China. Cell-line is a potential means used for long-term preservation of germplasm resources and an ideal in vitro model in place of living organisms for biological studies. Here, culture condition and characterization of fin-derived cell in Yangtze sturgeon were carried out. Tissue explant techniques have been efficiently used in the Yangtze sturgeon caudal fin (YSCF) culture. The YSCF cell line showed a fibroblast-like morphology and stable growth in minimum essential medium eagle's (MEME) supplemented with 10-20% fetal bovine serum at 25°C. Cells were cryopreserved with preservative DMSO in liquid nitrogen and grew normally after recovery. No bacterial, fungal, or mycoplasma contamination was detected in the YSCF cells. Karyotype analysis of the YSCF cells showed that the chromosome numbers of the YSCF ranged from 242 to 273, and the modal chromosome number was identified as 264 at passage 9. The YSCF cells were confirmed from A. dabryanus by assay of 16S rRNA and COI. Furthermore, GFP reporter gene was successfully transferred into YSCF cells and expressed. The established YSCF cell lines will contribute to the preservation of germplasm resources and provide a useful vitro tool for further biological studies in sturgeon species.

Establishment of Conditions for Long-Term Maintenance of Primary Embryonic Cell Cultures from Olive Flounder Paralichthys olivaceus.

Primary cell culture is a sufficient method frequently used to study the cellular properties and mechanisms of isolated cells in a controlled environment. In this study, an embryonic cell line (FGBC8) derived from the blastula stages of embryos of olive flounder Paralichthys olivaceus was developed. Furthermore, conditions for optimal long-term maintenance of this primary embryonic cell culture were investigated. Morphologically, FGBC8 cells were composed primarily of epithelial-like cells. FGBC8 cells were subcultured for >160 passages over ~830 days. The doubling time of FGBC8 cells was 73.8 h, and the modal diploid chromosome number was 48. FGBC8 cells transfected with green fluorescence protein (GFP)-expression plasmid exhibited a strong signal 48 h after transfection. Consequently, we demonstrated that fish serum is a crucial supplement for the long-term survival and maintenance of comparable morphology in these primary embryonic cells. Our results can be used as a guide for primary embryonic cell cultures for other fish species and may be useful for cell biotechnological applications.

Karyotype changes in long-term cultured tick cell lines

Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and other aspects of tick cellular processes. Tick cell cultures are often continuously cultivated, as freezing can affect their viability. However, the long-term cultivation of tick cells can influence their genome stability. In the present study, we investigated karyotype and genome size of tick cell lines. Though 16S rDNA sequencing showed the similarity between Ixodes spp. cell lines at different passages, their karyotypes differed from 2n = 28 chromosomes for parental Ixodes spp. ticks, and both increase and decrease in chromosome numbers were observed. For example, the highly passaged Ixodes scapularis cell line ISE18 and Ixodes ricinus cell lines IRE/CTVM19 and IRE/CTVM20 had modal chromosome numbers 48, 23 and 48, respectively. Also, the Ornithodoros moubata cell line OME/CTVM22 had the modal chromosome number 33 instead of 2n = 20 chromosomes for Ornithodoros spp. ticks. All studied tick cell lines had a larger genome size in comparison to the genomes of the parental ticks. Thus, highly passaged tick cell lines can be used for research purposes, but possible differences in encoded genetic information and downstream cellular processes, between different cell populations, should be taken into account.

Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2).

BackgroundHerpesviral hematopoietic necrosis disease, caused by cyprinid herpesvirus-2 (CyHV-2), is responsible for massive mortalities in the aquaculture of goldfish, Carassius auratus. Permissive cell lines for the isolation and propagation of CyHV-2 have been established from various goldfish tissues by sacrificing the fish. Here, we report the development of a cell line, FtGF (Fantail Goldfish Fin), from caudal fin of goldfish using non-lethal sampling. We also describe a simple protocol for successful establishment and characterization of a permissive cell line through explant method and continuous propagation of CyHV-2 with high viral titer using this cell line.MethodsCaudal fin tissue samples were collected from goldfish without killing the fish. Cell culture of goldfish caudal fin cells was carried out using Leibovitz’s L-15 (L-15) medium containing 20% FBS and 1X concentration of antibiotic antimycotic solution, incubated at 28 °C. Cells were characterized and origin of the cells was confirmed by sequencing fragments of the 16S rRNA and COI genes. CyHV-2 was grown in the FtGF cells and passaged continuously 20 times. The infectivity of the CyHV-2 isolated using FtGF cells was confirmed by experimental infection of naïve goldfish.ResultsThe cell line has been passaged up to 56 times in L-15 with 10% FBS. Karyotyping of FtGF cells at 30th, 40th and 56th passage indicated that modal chromosome number was 2n = 104. Species authentication of FtGF was performed by sequencing of the 16S rRNA and COI genes. The cell line was used for continuous propagation of CyHV-2 over 20 passages with high viral titer of 107.8±0.26 TCID50/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10th dpi. An experimental infection of naïve goldfish using supernatant from infected FtGF cells caused 100% mortality and CyHV-2 infection in the challenged fish was confirmed by the amplification of DNA polymerase gene, histopathology and transmission electron microscopy. These findings provide confirmation that the FtGF cell line is highly permissive to the propagation of CyHV-2.

Establishment and characterization of a new cell line from the muscle of humpback grouper (Cromileptes altivelis).

The humpback grouper (Cromileptes altivelis) is a commercially important species of the family Epinephelidae. With the development in aquaculture industry, C. altivelis breeding has gradually increased in volumetric production, leading to the occurrence of various diseases. In this study, we established a new cell line (CAM) derived from the muscle tissue of C. altivelis. Our results showed that the optimal growth temperature and working concentration of fetal bovine serum (FBS) of CAM cells were 28°C and 15%, respectively. DNA sequencing and comparative analysis of 18S rRNA gene sequence showed that CAM cell line was originated from C. altivelis. Chromosome analysis showed that the modal chromosome number of CAM cells was 48. After transfection using pEGFP-N3 plasmid, CAM cells exhibited high transfection efficiency, indicating that CAM cells could be used in foreign gene expression studies. Further, cytotoxicity analysis revealed that CAM cells were sensitive to Vibrio harveyi and Edwardsiella tarda. Moreover, the cytotoxicity of heavy metals (Hg, Cd, and Cu) to CAM cells was dose-dependent. This CAM cell line might be used as an ideal tool in vitro for analyzing and understanding the mechanisms of pathogenesis, host-pathogen interactions, and toxicity assay of heavy metals.

The Polyclonal Structure of Unisexual Prussian Carp (Carassius gibelio (Bloch, 1782)) Settlements in Northern Ukraine: Comparison Analysis of Allozyme Markers and Chromosome Numbers

An allozyme analysis of five sets of polymorphic loci of the gynogenetic Prussian carp C. gibelio from Northern Ukraine made it possible to identify within this species 29 genetic forms of clonal and recombinant nature, which can be divided into two categories. The first one is represented by the clonal C. gibelio-1 biotype with the modal number of 156 chromosomes and several unique alleles, which distinctly differentiate this biotype from the diploid goldfish C. auratus. The second category is represented by the group of 28 biotypes designated as C. gibelio-2 of a clonal and recombinant nature. Specimens of this group have no allozymes that distinguish them from C. auratus. Within this group, 158 and 160 modal chromosome groups were discovered; each one has special genotypic combination for locus Aat-1. Apparently, the series of biotypes, which are differentiated by the modal chromosome number, emerged independently as a result of hybridization of different pairs of parental species way back in Eastern Asia. At the same time, the emergence of recombinant individuals with unique combinations of genotypes of five protein loci and their own chromosome number within the C. gibelio-2 clone is a secondary event related to the implementation of the inheritance mechanism, which combines cloning and Mendelian inheritance. There are reasons to believe that this mechanism is implemented in pseudogamous crosses of genetically close C. gibelio-2 females with C. auratus males, while the cloning takes place in crosses of C. gibelio-2 females with more distant C. carassius males.

Susceptibility of betanodavirus in a newly established vertebra-derived cell line from Mosquitofish (Gambusia affinis).

In the present study, a new cell line from the vertebra of mosquitofish Gambusia affinis was successfully established and characterized. The cell line is named as bone Gambusia affinis (BGA) and subcultured for more than 55 passages in Leibovitz's/L15 medium supplemented with 15% FBS at 28°C. The cell line has a modal chromosome number of 48. Molecular characterization of the partial sequence of the coi gene confirmed the origin of the BGA cell line from mosquitofish. These cells exhibited epithelial morphology confirmed by the cytokeratin marker. The BGA cells showed mineralization of their extracellular matrix when stained with alizarin red and von Kossa stain. BGA cells were found to be susceptible to RGNNV and SJNNV strains of betanodavirus (NNV) showing cytopathic effect with multiple vacuolations in the cells. The RT-PCR confirmed the betanodavirus infections in BGA cells. The SEM micrograph showed the morphological changes observed in the cell during virus infection. The in vivo challenge experiment also showed the viral replicating efficiency in the Gambusia affinis with increasing viral titre. Thus, our present results show that the BGA cell line is a useful tool for isolating betanodavirus and could be used to investigate bone cell differentiation and extracellular matrix mineralization.

Virus susceptibility of a new cell line derived from the fin of black carp Mylopharyngodon piceus

A continuous cell line MPF derived from the fin of black carp Mylopharyngodon piceus was established and characterised in this study. Mylopharyngodon piceus fin (MPF) cells were subcultured for more than 80 passages with high viability recovery after long-term storage. The karyotyping analysis revealed that MPF had a modal diploid chromosome number (2n = 48) and identical ribosomal RNA sequence with black carp. In addition, the expression of pluripotency-associated markers including nanog, oct4 and vasa, were detected in MPF. The transient transfection efficiency of MPF reached 23% with a fluorescent reporter by modified electroporation and stable expression of red fluorescent MPF was established by the baculovirus system, indicating that MPF is an ideal platform for studying gene functions in vitro. Lastly, cytopathic effects were also observed and RNA transcripts of a viral gene increased after infection by spring viremia of carp virus (SVCV), suggesting that MPF could be an alternative tool for investigating pathogen-host interactions in black carp. In conclusion, a fin cell line that is susceptible to SVCV was established as a potential adult stem-cell line, providing a suitable tool for future genetic analyses and pathogen-host studies in black carp.

Karyotype changes in cultivated tick cell lines

Background. Tick cell lines are an easy-to-handle system for the study of viral and bacterial infections and different aspects of tick physiology. However, long-term cultivation of tick cells can influence genome stability. The aim of our work was to analyze and compare the karyotypes of Ixodes scapularis, I. ricinus, and Ornithodoros moubata tick cell lines after long-term been in culture. Methods. Mitotic spreads were prepared to count the number of chromosomes in ISE6, ISE18, IRE11, IRE/CTVM19, IRE/CTVM20, and OME/CTVM22 cell lines. The genome size of tick cell cultures was estimated by flow cytometry using propidium iodide staining. Results. The modal chromosome numbers around 22 and around 48 were typical for both I. ricinus and I. scapularis cell lines and differed from the diploid chromosome number in Ixodesticks – 28. In the OME/CTVM22cell line, the modal chromosome number was 33, instead of 20 in Ornithodoros ticks. All tick cell lines had a larger genome size in compare to genomes of parental ticks. Conclusions.Tick cell lines can be used for research purposes, however, differences in the internal processes between different cell populations should be taken into account. Acknowledgments: We thank Lesley Bell-Sakyi and the Tick Cell Biobank for provision of the tick cell lines.

Development and characterization of a skin cell line (SGA) from the mosquitofish Gambusia affinis and its susceptibility to fish Betanodavirus

A new continuous cell line designated as SGA has been developed from the skin tissue of the freshwater mosquitofish Gambusia affinis. The cell line grew well in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28 °C. Immunophenotyping of the cell line showed the epithelial nature of the cells. Chromosome number analysis showed that SGA cells have a modal diploid chromosome number of 48. Replication of the two different strains of betanodavirus (RGNNV and SJNNV) in SGA cell line showed the maximum virus titre of 108.82 TCID50 mL−1 for RGNNV and 107.2 TCID50 mL−1 for SJNNV. The cytopathic effect in the cell line was observed at 3 days post-infection (dpi) and multiple vacuolations were observed at 7 dpi. Further, DsRed2 plasmid was efficiently delivered into SGA cell line and was found that these cells were transfectable and can be used for assessing gene promoter activity. In vivo challenge experiments using the RGNNV infected cell culture supernatant showed signs of the disease in healthy mosquitofish with mortality commencing from 15 dpi. The above results suggest that the cell line is permissive for propagating betanodavirus and also could be an essential tool for studying the molecular pathogenesis of betanodavirus infection.

Single Nucleotide Polymorphism Array-Based Signature of Genetic Ploidy Groups in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is characterised by a number of recurrent chromosomal abnormalities which inform prognosis. Low hypodiploidy (HoTr) and high hyperdiploidy (HeH) are genetic subgroups associated with large non-random ploidy shifts, specifically 30-39 chromosomes and 51-65 chromosomes respectively. HoTr ALL often presents with a near triploid karyotype of 60-78 chromosomes through chromosomal endoreduplication without cytokinesis. This presents a diagnostic challenge in distinguishing this poor risk entity from good risk HeH ALL. To date, classification of such challenging cases has been based on the modal chromosome number, the pattern of specific gains, and identification of loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) arrays where possible. However, loss of cellular context and mixed cell populations (normal diploid, low hypodiploid and near-triploid) when analysing SNP arrays can pose additional analytical difficulty. The aim of this SNP array study was to (1) determine the level of inaccurate genetic subgrouping when cytogenetics only was used to distinguish HeH from doubled up HoTr and (2) develop a diagnostic algorithm to reliably call HeH and HoTr without supporting cytogenetic analysis. SNP arrays were performed on diagnostic ALL samples of 85 patients (46 HoTr, 39 HeH) using Illumina CytoSNP 850k (n=80) or Affymetrix Cytoscan HD (n=5) arrays. Probe level data were uploaded to Nexus Copy Number 10 (BioDiscovery) and manual segments spanning the length of each chromosome were created. No further data pre-processing was carried out and log2 ratio of 0 was automatically assigned to the median log2 ratio of the sample. Chromosomal log2 ratios were normalized within each sample and a variety of machine-learning techniques used to cluster the samples independently of assigned diagnosis. Cases residing in the incorrect cluster based on initial diagnosis were examined in detail using information from cytogenetics and SNP array interpretation. SNP arrays were analysed from 46 HoTr (median age 50.5 years (range 7-87), 43% male) and 39 HeH (median age 7 years (range 1-58), 56% male) patients. Unsupervised clustering of log2 ratios showed a clear distinction between HeH and HoTr patients (figure (A)). Six cases clustered incorrectly based on cytogenetic diagnosis. After detailed interpretation of all cases, including identifying LOH affecting chromosomes 3, 7, 15, 16 and 17, 3/6 cases initially classified as HeH were highly suggestive of HoTr ALL despite having <60 chromosomes. Similarly, 1/6 cases was cytogenetically diagnosed with HoTr but had a SNP array pattern typical of HeH ALL. We identified chromosomes 1, 4, 11, 17, 19, and 21 as those contributing most to the distinction in the HoTr and HeH signatures with the log2 ratio of chromosome 1 the most highly discriminatory in this cohort. Using whole chromosome log2 ratios, HoTr and HeH ALL have distinct profiles. SNP array analysis highlighted at least 4 patients whose ploidy subgroups appeared incorrectly called by cytogenetics, which can affect risk stratification. Crucially, these data call into question the accepted modal chromosome numbers for HeH and HoTr in the near triploid phase and suggest this alone cannot be used to classify patients into these ploidy groups. All samples incorrectly classified as HeH ALL were from adults aged >40 years, suggesting this good risk subgroup is even rarer than previously thought in older adults. After re-classification, our cohort only contained 5/41 adults >40 years with HeH ALL, signifying that HoTr ALL is the commonest genetic ploidy group in older adults with ALL and must still be considered in cases with 50-60 chromosomes. Copy number analysis from SNP arrays is challenging in samples with marked ploidy shift and mixed cell populations. Importantly, a number of our samples had significant contaminating normal DNA and LOH could not be confirmed visually (figure (B)), underlying the need for additional factors to aid classification. Our analysis only takes into account log2 ratio of entire chromosomes, thus permitting a measure of the relative over and under-representation of specific chromosomes within the sample. This method accurately clusters patients even when LOH cannot be clearly visualized from B-allele frequency due to contaminating non-leukemic DNA and supports the development of a diagnostic classifier based on chromosomal log2 ratios. Disclosures Fielding: Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy.

Establishment and characterization of a fin tissue cell line derived from silver pomfret, Pampus argenteus.

A cell line (PaF) derived from the fin tissue of silver pomfret (Pampus argenteus) was established and characterized in this study. The cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium (DMEM) containing 15% foetal bovine serum (FBS) since the initial primary culture. PaF cells grew well at temperatures from 24°C to 28°C in DMEM supplemented with 15% FBS. Partial amplification and sequence analysis of the cytochrome B gene indicated that PaF originated from silver pomfret. Cytogenetic analysis demonstrated that the modal chromosome number was 48. A significant cytopathic effect was observed in PaF cells during viral haemorrhagic septicaemia virus (VHSV) infection, and the VHSV replication was confirmed by qRT-PCR and viral titre assays. In contrast, PaF cells were resistant to red-spotted grouper nervous necrosis virus infection. Moreover, PaF cells could respond to VHSV and lipopolysaccharide treatments, as indicated by the expression of immune-related genes, TLR5 and TLR9. In conclusion, the establishment of PaF cell line will provide an appropriate in vitro tool for the study of mechanisms of pathogen-silver pomfret interaction.

Microarray testing as an efficient tool to redefine hyperdiploid paediatric B-cell precursor acute lymphoblastic leukaemia patients

The aim of our study was to characterize genetic alterations in a cohort of paediatric patients with B-cell progenitors (BCP-ALL) and a hyperdiploid karyotype. In our study, we analysed 55 childhood hyperdiploid BCP-ALL patients using single nucleotide polymorphism (SNP) microarray testing. The group consisted mostly of patients with the modal number of chromosomes between 54 and 58 (34 cases). Within this group, Trisomy 4 and Trisomy 10 (30 cases) were the most frequent cases. Additionally, a total of 93 structural abnormalities mainly affecting chromosomes 1, 6, 9, 12, and 17 as well as 68 copy number alterations (CNAs) were identified. The microarray testing revealed a loss of ETV6, IKZF1, CDKN2A/CDKN2B, PAX5, and RB1. Moreover, chromosomal abnormalities resulting in the loss of heterozygosity (LOH) were also observed. Currently, patients with hyperdiploidy constitute a genetically heterogeneous group, and therefore, it is insufficient to rely only on banding cytogenetic analysis for the identification of hyperdiploid karyotype. Microarray testing has been proven an effective and satisfactory tool for the analysis of molecular karyotypes and to redefine the prognostic criteria in hyperdiploid patients.

Establishment and characterization of a novel cell line (cc‑006cpm8) of moderately/poorly differentiated colorectal adenocarcinoma derived from a primary tumor of a patient.

In the present study, the cc‑006cpm8 novel colon cell line was established from a sample of right colorectal adenocarcinoma obtained from a woman with liver metastasis. It was possible to culture this cell line for ≥100passages invitro with vigorous growth. Morphologically, the cells grew as several layers with tight adhesion to the surface of the culture plate. The morphological, immunological and ultrastructural features of these cells suggested their epithelial origin. The characterization of this cell line indicated a doubling time of 27h, a colony forming efficiency of 73.2% in semisolid media and a plate efficiency of 66.5% in liquid culture. The modal number of chromosomes was50. Invivo, the cc‑006cpm8 cells underwent tumorigenesis in all nude mice used. Immunohistochemical analysis demonstrated that mutS homolog2(MSH2) and MSH6 were expressed; however, mutL homolog1 and postmeiotic segregation2 were downregulated in cc‑006cpm8 cells. To determine the mutation profile of the cell line analyzed, exome capture DNA sequencing was performed. The results revealed 20hypermutated exons comprising single nucleotide polymorphisms, and insertion and deletions(InDels), including single nucleotide variants of mucin(MUC)19, MUC16, MUC12, filaggrin and AHNAK nucleoprotein2, and InDels of βdefensin‑126, microRNA‑3665, WNK lysine deficient protein kinase1 and SLAIN motif‑containing protein1. In addition, commonly mutated genes in colorectal cancer and exon mutations of genes in cc‑006cpm8 cells were analyzed, including adenomatous polyposis coli, tumor protein p53, Drosophila mothers against decapentaplegic4, phosphatidylinositol‑4,5‑bisphosphate3‑kinase catalytic subunitα and Kirsten rat sarcoma, and genes associated with the DNA mismatch repair pathway were investigated.

Establishment and characterization of a cell line from tilapia brain for detection of tilapia lake virus.

Tilapia lake virus (TiLV) is an emerging disease threatening tilapia culture in many parts of the world. A cell line from the brain of tilapia, which was named TiB, was established, characterized and subcultured with more than 100 passages. The TiB cell line was optimally maintained at 27°C using medium 199 (M199) supplemented with 10% foetal bovine serum (FBS). Chromosome analysis revealed that 60% of TiB cells at passage 5 maintained the modal chromosome number 2n=44, while at passage 60, there were 43% of TiB cells with the diploid chromosome number 2n=50. A significant cytopathic effect was observed in TiB cells after infection with tilapia lake virus (TiLV-2017A), and the viral replication in the cells was confirmed by transmission electron microscopy, immunofluorescence assays and viral titres, indicating the susceptibility of TiB cells to TiLV-2017A. The viral titres of TiLV-2017A in TiB cells reached 107.43 TCID50 /ml within 10days. The stable growth and susceptibility to fish viruses make TiB cells a useful tool for fish virus-host cell interaction and for immune response of fish.

Development and Characterization of a New Cell Line from Olive Flounder Paralichthys olivaceus.

A new embryonic cell line (OFEC-17FEN) derived from olive flounder Paralichthys olivaceus was developed. OFEC-17FEN cells were subcultured for <30 passages over ~200 days. OFEC-17FEN cells had a doubling time of 114.34 h and modal diploid chromosome number was 48. The pluripotency genes POU5f1 and NANOG were expressed in OFEC-17FEN cells. However, the lack of several pluripotency-related genes expression indicates that OFEC-17FEN cells are not stem cells. OFEC-17FEN cells transfected with plasmid pEGFP-c1 exhibited a strong green fluorescent signal at 48 h after transfection. Accordingly, OFEC-17FEN cells may be useful for both basic research and biotechnological application.

Establishment and characterization of a caudal fin-derived cell line, AOF, from the Oscar, Astronotus ocellatus.

Astronotus ocellatus, commonly called the oscar, is one of the popular cichlids among aquarium hobby. The present study deals with the development and characterization of a new cell line from caudal fin of A. ocellatus. The cell line was cultured in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28°C. The optimum temperature and FBS concentration for cell growth were tested with temperature ranges from 20 to 37°C and FBS concentrations of 5-20% at 28°C. The Astronotus ocellatus fin cell line has been subcultured 45 times since its development and the modal chromosome number (2n) is 48. The cell line is composed mainly of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies. The cell line was cryopreserved at different passage levels and the revival efficiency showed 80% survival rate. Partial sequence amplification and sequencing of two genes, mitochondrial 16S ribosomal RNA and cytochrome oxidase I, confirmed the origin of cell line. The cell line did not show Mycoplasma contamination. The cells showed good transfection efficiency when transfected with 2μg of pAcGFP1-N1 expression vector. The extracellular products of fish bacterial pathogens viz., Aeromonas hydrophila and A. caviae, were cytotoxic to AOF cells but were not susceptible to Cyprinid herpes virus 2. The development of AOF cell line will have significant applications in fish virology and will prove useful to isolate pathogens in the event of sudden viral disease outbreak and for the development of vaccines and diagnostic kits.

Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5.

Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations and a reduced modal chromosomal number compared to RPMI-8402. soaCGH analysis identified vast copy number alterations and >200 unbalanced DNA breakpoints distributed unevenly across the chromosomal complement in CPT-K5. In addition, the short tandem repeat alleles were found to be highly different between CPT-K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT.