Articles de revues : « Trout-derived cell lines »

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Mellanen, Pirkko, Tiina Petänen, Jyrki Lehtimäki, Sari Mäkelä, Göran Bylund, Bjarne Holmbom, Erkki Mannila, Aimo Oikari et Risto Santti. « Wood-Derived Estrogens : Studiesin Vitrowith Breast Cancer Cell Lines andin Vivoin Trout ». Toxicology and Applied Pharmacology 136, n^o 2 (février 1996) : 381–88. http://dx.doi.org/10.1006/taap.1996.0046.

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Gratacap, Remi L., Ye Hwa Jin, Marina Mantsopoulou et Ross D. Houston. « Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes ». Marine Biotechnology 22, n^o 5 (18 septembre 2020) : 717–24. http://dx.doi.org/10.1007/s10126-020-09995-y.

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Abstract Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

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Chen, Maria J., Pinwen Peter Chiou, Yu-Hsian Liao, Chun-Mean Lin et Thomas T. Chen. « Development and characterization of five rainbow trout pituitary single-cell clone lines capable of producing pituitary hormones ». Journal of Endocrinology 205, n^o 1 (19 janvier 2010) : 69–78. http://dx.doi.org/10.1677/joe-09-0315.

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Five single-cell clone lines (mRTP1B, mRTP1E, mRTP1F, mRTP1K, and mRTP2A) have been developed from adult rainbow trout pituitary glands. These cell lines have been maintained in a CO2-independent medium supplemented with 10% fetal bovine serum (FBS) for more than 150 passages. At about 150 passages, the doubling time of each single-cell clone in a CO2-independent medium supplemented with 10% FBS at 20 °C was 3.6±0.7, 2.8±0.7, 3.2±0.8, 5.5±0.6, and 6.6±0.6 days respectively. Each single-cell clone contains 60±2 chromosomes, which is within the range of the 2N chromosome numbers reported for rainbow trout. Reverse transcription-PCR analysis revealed that in addition to expressing gh, prolactin (prl), and estradiol (E2) receptor α (e2rα or esr1) genes, each single-cell clone line also expressed other pituitary-specific genes such as tsh, gonadotropin 1 (gth-1 or fshb), gonadotropin 2 (gth-2 or lhb), somatolactin (sl or smtl), proopiomelanocortin-B (pomcb), and corticosteroid receptor (cr or nr3c1). Immunocytochemical analysis showed that all the five single-cell clones produced both Gh and Prl. Furthermore, the expression of gh and prl genes in the single-cell clone lines is responsive to induction by E2, dexamethasone, and o,p′-dichlorodiphenyltrichloroethane. All together, these results confirm that each of the single-cell clones was derived from rainbow trout pituitary glands. These single-cell clone lines not only can be used to study factors that regulate the expression of pituitary hormone genes, but can also be developed as a rapid screening system for identifying environmental endocrine disruptors.

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Pasquariello, Rolando, Nicole Verdile, Radmila Pavlovic, Sara Panseri, Kristin Schirmer, Tiziana A. L. Brevini et Fulvio Gandolfi. « New Stable Cell Lines Derived from the Proximal and Distal Intestine of Rainbow Trout (Oncorhynchus mykiss) Retain Several Properties Observed In Vivo ». Cells 10, n^o 6 (19 juin 2021) : 1555. http://dx.doi.org/10.3390/cells10061555.

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We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose. High seeding density induced their differentiation into more mature phenotypes, as indicated by the downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5), whereas alkaline phosphatase activity was upregulated. Other enterocyte markers (i.e., sglt1 and pept1), however, were not regulated as expected. In all cell lines, the presence of a mixed population of epithelial and stromal cells was characterized for the first time. The expression by the stromal component of lgr5, a stem cell niche regulatory molecule, may explain why these lines proliferate stably in vitro. Although most parameters were conserved among the three cell lines, some significant differences were observed, suggesting that characteristics typical of each tract are partly conserved in vitro as well.

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Ito, T., et T. Kamaishi. « Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major susceptibility to infectious hematopoietic necrosis virus (IHNV) isolate ». Diseases of Aquatic Organisms 146 (12 août 2021) : 1–8. http://dx.doi.org/10.3354/dao03615.

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Infectious hematopoietic necrosis virus (IHNV) is known as a causative agent of heavy mortalities in farmed rainbow trout. However, there is limited information on its virulence for marine fish species. In this study, Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major were experimentally infected by intraperitoneal (IP) injection and immersion, with an IHNV isolate from rainbow trout in Japan, to evaluate the virulence of the virus for these fish species. The cumulative mortality for immersed rainbow trout was 15%. IHNV was isolated from all dead fish and 50% of the sequentially sampled rainbow trout. When Japanese amberjack were challenged by IP injection and immersion, the resulting cumulative mortality was 70% and 0%, respectively. The virus was isolated from all dead fish and 1 out of 3 Japanese amberjack sampled at 9 d post exposure. However, no mortality was observed in either of the red sea bream groups challenged with IHNV. IHNV was not isolated from any of the surviving red sea bream, or from any of the sequentially sampled fish. The viral titer on Japanese amberjack-derived YTF cells was in the same log range as that on FHM and RTH-149 cells, but the titers on the red sea bream cell lines SBK and GBRS were lower than the other cell lines, and were significantly different from the FHM and RTH-149 cell lines. These results suggest that Japanese amberjack has a low susceptibility to IHNV, and red sea bream has no or little susceptibility to the virus.

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Bearzotti, Monique, Bernard Delmas, Annie Lamoureux, Anne-Marie Loustau, Stefan Chilmonczyk et Michel Bremont. « Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin ». Journal of Virology 73, n^o 9 (1 septembre 1999) : 7703–9. http://dx.doi.org/10.1128/jvi.73.9.7703-7709.1999.

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ABSTRACT Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to theRhabdoviridae family.

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Clemons, J. H., M. R. van den Heuvel, J. J. Stegeman, D. G. Dixon et N. C. Bols. « Comparison of Toxic Equivalent Factors for Selected Dioxin and Furan Congeners Derived Using Fish and Mammalian Liver Cell Lines ». Canadian Journal of Fisheries and Aquatic Sciences 51, n^o 7 (1 juillet 1994) : 1577–84. http://dx.doi.org/10.1139/f94-156.

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Toxic equivalent factors (TEFs) for eight polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) congeners were derived with a rainbow trout (Oncorhynchus mykiss) cell line, RTL-W1, and compared with TEFs obtained with a rat hepatoma cell line, H4IIE. Cells were exposed to a range of concentrations of the congeners which included 1,2,3,7,8-pentaCDD, 1,2,3,4,7,8-hexaCDD, 1,2,3,6,7,8-hexaCDD, 1,2,3,4,6,7,8-heptaCDD, 2,3,7,8-tetraCDF, 1,2,3,7,8-pentaCDF, 2,3,4,7,8-pentaCDF, and 1,2,3,4,7,8-hexaCDF. Ethoxyresorufin o-deethylase (EROD) activity was measured and EC50 values calculated from a dose–effect curve newly proposed for this purpose. TEFs were computed using 2,3,7,8-tetraCDD standard curves run concurrently with each assay. With the exception of 1,2,3,6,7,8-hexaCDD and 1,2,3,7,8-pentaCDF, all of the RTL-W1-derived TEFs were significantly higher (two- to eightfold higher) than the respective H4IIE TEFs. Immunoblotting analysis with the monoclonal anti-scup P4501A1 antibody was used to identify basal and induced levels of P4501A1 protein in both the RTL-W1 and H4IIE cells. It was concluded that mammalian-derived TEFs may not accurately predict the potency of PCDDs or PCDFs to fish.

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Boudinot, Pierre, Sabine Riffault, Samia Salhi, Charles Carrat, Christine Sedlik, Nassira Mahmoudi, Bernard Charley et Abdenour Benmansour. « Vesicular stomatitis virus and pseudorabies virus induce a vig1/cig5 homologue in mouse dendritic cells via different pathways ». Journal of General Virology 81, n^o 11 (1 novembre 2000) : 2675–82. http://dx.doi.org/10.1099/0022-1317-81-11-2675.

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The homologous genes vig1 and cig5 were identified by differential display PCR as virus-induced genes in rainbow trout and humans, respectively. These genes are significantly related to sequences required for the biosynthesis of metal cofactors, but their function remains unknown. In this study, it is shown that the mouse homologue of vig1/cig5 was induced by vesicular stomatitis virus (VSV) and pseudorabies virus (PrV) in mouse spleen cells. Among a collection of cell lines from dendritic, myeloid, lymphoid or fibroblast lineages, only the dendritic cell line, D2SC1, showed expression of mvig after virus infection. This dendritic restriction was confirmed by our finding that mvig was also induced by both VSV and PrV in CD11c++ spleen cells, separated by magnetic purification or derived from bone marrow precursor cells. Similar to the fish rhabdovirus viral haemorrhagic septicaemia virus in trout cells, VSV directly induced mvig in the dendritic cell line D2SC1, but the PrV-mediated induction required the integrity of the interferon pathway. This result indicates that mvig is interferon-inducible like its fish and human homologues. Furthermore, mvig was also induced by LPS in bone marrow-derived cells. Thus, mvig expression seems to correlate with an activated state of dendritic cells subjected to different pathogen-associated stimuli.

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Carter, Charleata A., William W. Ellington et Rebecca J. Van Beneden. « Confocal Laser Scanning Microscopy of Oncogene Localization in Rainbow Trout Cell Lines Derived from Normal and Tumor Tissue*1 ». Toxicologic Pathology 24, n^o 3 (mai 1996) : 339–45. http://dx.doi.org/10.1177/019262339602400310.

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Pinel, Karine, Cécile Heraud, Guillaume Morin, Karine Dias, Annaëlle Marcé, Linda Beauclair, Stéphanie Fontagné-Dicharry et al. « Are the Main Methionine Sources Equivalent ? A Focus on DL-Methionine and DL-Methionine Hydroxy Analog Reveals Differences on Rainbow Trout Hepatic Cell Lines Functions ». International Journal of Molecular Sciences 23, n^o 6 (8 mars 2022) : 2935. http://dx.doi.org/10.3390/ijms23062935.

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The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the metabolic needs of fish of agronomic interest such as rainbow trout (RT-Oncorhynchus mykiss). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.

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Hassan, Intisar A., Zachary Renfro, Harrison Blake, Satyajit Rath et Jeannine M. Durdik. « Effect of temperature on functional activity of macrophages in three different species ». Journal of Immunology 204, n^o 1_Supplement (1 mai 2020) : 149.17. http://dx.doi.org/10.4049/jimmunol.204.supp.149.17.

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Abstract Temperature affects body physiological functions, including immunity in ways that influence survival. This study investigated the effect of temperature on macrophage activation and metabolism across species. Macrophages were isolated from mice, chicken or rainbow trout primary tissue or from macrophage cell lines and were activated with lipopolysaccharide (LPS) or IFN-γ. For mice, both Raw264.7 and primary cells, nitric oxide (NO) production was similar at 35°C and 37°C but dropped dramatically at temperatures below 35°C. At fever temperature (39°C), NO release increased in response to LPS. Young bone marrow derived macrophage (BMDM) and peritoneal resident macrophage (PRM) showed increased protein synthesis at 39°C compared to 37°C. Chicken splenic macrophages (CSM) showed NO responses that were similar at 37, 39, and at 41°C (normal for avian). A fever of 42°C had a large stimulatory effect on NO production compared to 41°C. A chicken liver derived macrophage cell line, HTC, showed the same pattern. They also showed higher protein synthesis at 42°C compared with (41°C) after LPS stimulation. Trout head-kidney macrophages (THM) showed the highest NO responses at 19°C when compared to more typical stream temperatures of 13, 15, and 16°C. They still showed some response at 28 and 37°C. Thus, their macrophages respond at higher temperatures than the fish can tolerate. Stimulated THM cells with LPS at 19°C showed increased protein synthesis compared to 13 and 16°C. We can conclude from our experiments that fish macrophages had a much broader range of temperatures at which they could respond by NO generation and protein synthesis compared to mice and chickens and all had increased responses at fever temperatures.

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Fischer, Stephan, Jovica Loncar, Roko Zaja, Sabine Schnell, Kristin Schirmer, Tvrtko Smital et Till Luckenbach. « Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss) ». Aquatic Toxicology 101, n^o 2 (janvier 2011) : 438–46. http://dx.doi.org/10.1016/j.aquatox.2010.11.010.

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Villoing, Stéphane, Monique Béarzotti, Stefan Chilmonczyk, Jeannette Castric et Michel Brémont. « Rainbow Trout Sleeping Disease Virus Is an Atypical Alphavirus ». Journal of Virology 74, n^o 1 (1 janvier 2000) : 173–83. http://dx.doi.org/10.1128/jvi.74.1.173-183.2000.

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ABSTRACT Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10°C, while little virus was produced at 14°C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.

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Yeung, Chung-Man, Chi-Bun Chan, Norman Y. S. Woo et Christopher H. K. Cheng. « Seabream ghrelin : cDNA cloning, genomic organization and promoter studies ». Journal of Endocrinology 189, n^o 2 (mai 2006) : 365–79. http://dx.doi.org/10.1677/joe.1.06593.

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Recent studies have indicated that ghrelin stimulates growth hormone release from the pituitary via the growth hormone secretagogue receptor (GHSR). We have previously isolated two GHSR subtypes from the pituitary of the black seabream Acanthopagrus schlegeli. In the present study, we have cloned and characterized ghrelin from the same fish species at both the cDNA and gene levels. The full-length seabream ghrelin cDNA, isolated from sea-bream stomach using a novel approach by exploiting a single conserved region in the coding region, was found to encode a prepropeptide of 107 amino acids, with the predicted mature ghrelin peptide consisting of 20 amino acids (GSSFLSPSQKPQNRGKSSRV). Embedded in this full-length cDNA is a putative fish orthologue of the recently reported mammalian obestatin peptide. The ghrelin gene in black seabream, obtained by genomic PCR, was found to encompass four exons and three introns, possessing the same structural organization as in tilapia and goldfish, but different from that in rainbow trout. In addition, a 2230-bp 5′-flanking region of the seabream ghrelin gene was obtained by genome walking. Sequence analysis revealed that, as in the case of the human ghrelin gene, there is neither a GC box nor a CAAT box present in the isolated 5′-flanking region. However, a number of putative transcription factor-binding sites different from the human counterpart were found in the 5′-flanking region of the seabream ghrelin gene, suggesting that different cis- and trans-acting elements are involved in controlling their gene expression. Functional activity of this 5′-flanking region was examined by cloning it into the pGL3-Basic vector upstream of the luciferase reporter gene and transfected into various cell lines. Positive promoter activity could only be recorded in the colon-derived Caco-2 cells, suggesting that the cloned 5′-flanking region represents the functional promoter of the seabream ghrelin gene, which exhibits tissue-specific promoter activity. Using reverse transcriptase PCR analysis, expression of ghrelin was detected only in the seabream stomach, but not in the other tissues examined, including the brain, gill, intestine, kidney, liver and spleen. This stomach-specific expression of ghrelin in seabream is subject to regulation, as administration of growth hormone or ipamorelin to the fish in vivo was demonstrated to enhance its expression. Reminiscent of the homologous upregulation found in the transcriptional control of the seabream GHSR gene, a similar homologous regulatory mechanism might also exist in controlling the expression of seabream ghrelin. The identification of both GHSR and ghrelin from a single fish species would facilitate our subsequent studies on the elucidation of the physiological functions of the ghrelin/GHSR system in teleost. The possible existence of obestatin in teleost opens up new research avenues on the somatotropic axis in fish.

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Zoppo, Marina, Nicole Okoniewski, Stanislav Pantelyushin, Johannes vom Berg et Kristin Schirmer. « A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells ». Cell & ; Bioscience 11, n^o 1 (3 juin 2021). http://dx.doi.org/10.1186/s13578-021-00618-0.

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Abstract Background The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. Results Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. Conclusions The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

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