scholarly journals Plasma Treatment of Fish Cells: The Importance of Defining Cell Culture Conditions in Comparative Studies

2021 ◽  
Vol 11 (6) ◽  
pp. 2534
Author(s):  
Henrike Rebl ◽  
Claudia Bergemann ◽  
Sebastian Rakers ◽  
Barbara Nebe ◽  
Alexander Rebl
Keyword(s):  
Plasma Treatment ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Atmospheric Pressure Plasma ◽  
Fish Farming ◽  
Skin Lesions ◽  
Atmospheric Plasma ◽  
Fish Cell ◽  
Farmed Fish ◽  
Fish Cells

The present study provides the fundamental results for the treatment of marine organisms with cold atmospheric pressure plasma. In farmed fish, skin lesions may occur as a result of intensive fish farming. Cold atmospheric plasma offers promising medical potential in wound healing processes. Since the underlying plasma-mediated mechanisms at the physical and cellular level are yet to be fully understood, we investigated the sensitivity of three fish cell lines to plasma treatment in comparison with mammalian cells. We varied (I) cell density, (II) culture medium, and (III) pyruvate concentration in the medium as experimental parameters. Depending on the experimental setup, the plasma treatment affected the viability of the different cell lines to varying degrees. We conclude that it is mandatory to use similar cell densities and an identical medium, or at least a medium with identical antioxidant capacity, when studying plasma effects on different cell lines. Altogether, fish cells showed a higher sensitivity towards plasma treatment than mammalian cells in most of our setups. These results should increase the understanding of the future treatment of fish.

scholarly journals Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 75 ◽  
Author(s):  
Sebastian Escobar-Aguirre ◽  
Duxan Arancibia ◽  
Amanda Escorza ◽  
Cristián Bravo ◽  
María Andrés ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Lines ◽  
Gene Function ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
Guide Rna ◽  
Bicistronic Vector ◽  
Expression Platform ◽  
Fish Cells ◽  
U6 Rna

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function.

An Efficient Vector-based CRISPR/Cas9 System in an Oreochromis mossambicus Cell Line using Endogenous Promoters

2020 ◽  
Author(s):  
Jens Hamar ◽  
Dietmar Kültz
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Gene Editing ◽  
Ectopic Expression ◽  
Brain Cell ◽  
Genomic Alteration ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
Fish Cells ◽  
Cellular Phenotypes

AbstractCRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmbAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

scholarly journals Cold Atmospheric Pressure Plasma Treatment Modulates Human Monocytes/Macrophages Responsiveness

Plasma ◽  
2018 ◽  
Vol 1 (2) ◽  
pp. 261-276 ◽  
Author(s):  
Letizia Crestale ◽  
Romolo Laurita ◽  
Anna Liguori ◽  
Augusto Stancampiano ◽  
Maria Talmon ◽  
...  
Keyword(s):  
Plasma Treatment ◽  
Macrophage Polarization ◽  
Immune Surveillance ◽  
Atmospheric Pressure Plasma ◽  
Atmospheric Plasma ◽  
Viability Analysis ◽  
Inflammatory Phenotype ◽  
Cold Atmospheric Pressure Plasma ◽  
Anti Inflammatory ◽  
Inflammatory Macrophages

Monocytes are involved in innate immune surveillance, establishment and resolution on inflammation, and can polarize versus M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. The possibility to control and drive immune cells activity through plasma stimulation is therefore attractive. We focused on the effects induced by cold-atmospheric plasma on human primary monocytes and monocyte-derived macrophages. Monocytes resulted more susceptible than monocyte-derived macrophages to the plasma treatment as demonstrated by the increase in reactive oxygen (ROS) production and reduction of viability. Macrophages instead were not induced to produce ROS and presented a stable viability. Analysis of macrophage markers demonstrated a time-dependent decrease of the M1 population and a correspondent increase of M2 monocyte-derived macrophages (MDM). These findings suggest that plasma treatment may drive macrophage polarization towards an anti-inflammatory phenotype.

Cytotoxicity evaluation of chemicals using cultured fish cells

2000 ◽  
Vol 42 (7-8) ◽  
pp. 277-282 ◽  
Author(s):  
M. Mori ◽  
M. Wakabayashi
Keyword(s):  
Cell Lines ◽  
Copper Chloride ◽  
Nickel Chloride ◽  
Neutral Red ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
Neutral Red Assay ◽  
Cultured Fish ◽  
Fish Cells ◽  
Very High

Development of simple and rapid cytotoxicity assays using fish cell lines was attempted. Cytotoxicity of chemicals was evaluated by neutral red assay using five monolayer-cultured fish cell lines, BB cells, CHSE-214 cells, EPC cells, FHM cells, RTG-2 cells, and one suspension-cultured fish cell line, CHSE-sp cells. The strength of cytotoxicity was observed in an order of cadmium chloride>zinc sulfate>copper chloride>nickel chloride. The concentration of no observable effect on the CHSE-214 cells viability was 2%(v/v) for DMSO and 0.5%(v/v) for DMSO containing 20 mg · ml–1 taurodeoxycholic acid. The correlation between the 24-hour NR50 values of eleven chemicals to the CHSE-sp cells and those to the CHSE-214 cells was very high (r=0.98). The cytotoxicity assay using suspension-cultured fish cells was found to be one of valuable device for screening the toxicity of chemicals for fish.

Host cell invasion and intracellular residence byAeromonas salmonicida: Role of the S-layer

2000 ◽  
Vol 46 (7) ◽  
pp. 660-668 ◽  
Author(s):  
Rafael A Garduño ◽  
Anne R Moore ◽  
Gilles Olivier ◽  
Angela L Lizama ◽  
Elizabeth Garduño ◽  
...  
Keyword(s):  
Cell Lines ◽  
Layer Structure ◽  
Aeromonas Salmonicida ◽  
Fish Cell ◽  
Cell Infection ◽  
Fish Cell Lines ◽  
Invasion Mechanisms ◽  
Cell Counts ◽  
Latex Beads ◽  
Fish Cells

Virulent strains of the fish pathogen Aeromonas salmonicida, which have surface S-layers (S+), efficiently adhere to, enter, and survive within macrophages. Here we report that S+bacteria were 10- to 20-fold more adherent to non-phagocytic fish cell lines than S-layer-negative (S-) mutants. When reconstituted with exogenous S-layers, these S-mutants regained adherence. As well, latex beads coated with purified S-layers were more adherent to fish cell lines than uncoated beads, or beads coated with disorganized S-layers, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure. Gentamicin protection assays and electron microscopy indicated that both S+and S-A. salmonicida invaded non-phagocytic fish cells. In addition, these fish cells were unable to internalize S-layer-coated beads, clearly suggesting that the S-layer is not an invasion factor. Lipopolysaccharide (which is partially exposed in S+bacteria) appeared to mediate invasion. Surprisingly, A. salmonicida did not show net growth inside fish cells cultured in the presence of gentamicin, as determined by viable bacterial cell counts. On the contrary, bacterial viability sharply decreased after cell infection. We thus concluded that the S-layer is an adhesin that promotes but does not mediate invasion of non-phagocytic fish cell lines. These cell lines should prove useful in studies aimed at characterizing the invasion mechanisms of A. salmonicida, but of limited value in studying the intracellular residence and replication of this invasive bacterium in vitro.Key words: Aeromonas salmonicida, invasion, S-layer, lipopolysaccharide, capsule, intracellular bacteria, furunculosis.

scholarly journals An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jens Hamar ◽  
Dietmar Kültz
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Gene Editing ◽  
Ectopic Expression ◽  
Brain Cell ◽  
Genomic Alteration ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
Fish Cells ◽  
Cellular Phenotypes

AbstractCRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

scholarly journals Assessment of engineered nanosilver as an alternative nano-antibiotic in marine water pollution using biomarker of fish cell line

2021 ◽  
Vol 5 ◽  
pp. 239784732199828
Author(s):  
Tayyaba BiBi ◽  
Taj Muhammad Khan
Keyword(s):  
Cell Lines ◽  
Fish Cell ◽  
Fish Farms ◽  
Fish Cell Lines ◽  
Farmed Fish ◽  
Resistant Genes ◽  
Frequent Use ◽  
Animal Pathogens ◽  
Ocean Environment ◽  
Complex Scenario

A large volume of antibiotics is used in fish farms to treat diseases because the farmed fish are fully affected by diseases and parasites in the aquaculture and particularly in the ocean environment where disease pathogens multiply quickly. The frequent use of these antibiotics in aquaculture has resulted in animal; stress, infection, and their dissemination in the form of antibiotic resistant genes to other bacteria including human and animal pathogens. The problems arising with antibiotics can be overcome by using silver nanoparticles (AgNPs) due to their physiochemical properties and low toxicity. So AgNPs could be combined with antibiotics to induce infections in fish cell lines and to protect dissemination of antibiotics in the form of antibiotics resistant genes. We expose AgNPs on fish cell lines as a new nano-antibacterial agent to investigate and obtain findings in terms of the cell viability and toxicity. The experimental data is analyzed to examine the antibacterial effects of nanosilver as a replacement agent and discuss the complex scenario, drawbacks, techniques, methods, main mechanisms, and procedures to perform antibacterial tests of exposed AgNPs. There would be an attempt to deal with the AgNPs antibacterial therapies for the fish cell lines.

scholarly journals DNA replication and repair in Tilapia cells. I. The effect of ultraviolet radiation

1984 ◽  
Vol 72 (1) ◽  
pp. 213-226
Author(s):  
F.H. Yew ◽  
L.M. Chang
Keyword(s):  
Ultraviolet Radiation ◽  
Dna Synthesis ◽  
Cell Survival ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Fish Cell ◽  
Dna Replication And Repair ◽  
Post Replication Repair ◽  
A Cell

The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor.

Wettability Control of VACNT Array through Atmospheric Plasma Treatment

2015 ◽  
Vol 137 (2) ◽  
Author(s):  
Yulong Ji ◽  
Gen Li ◽  
Yuqing Sun
Keyword(s):  
Contact Angle ◽  
Plasma Treatment ◽  
Atmospheric Pressure ◽  
Atmospheric Pressure Plasma ◽  
Plasma Jet ◽  
Contact Angle Measurement ◽  
Angle Measurement ◽  
Atmospheric Plasma ◽  
Drop Shape ◽  
Vertically Aligned

Wetting characteristics of the vertically aligned carbon nanotube (VACNT) array surface can be modified by atmospheric plasma treatment with masks. The plasma treatment of the VACNT array surface was performed by moving an atmospheric pressure plasma jet system (Plasmatreat GmbH, Steinhagen) at a fixed speed of 0.167 m/s through a mask with holes. The hole diameter is fixed equal to 250 µm. The distance between holes varies between 0.5 mm to 3.0 mm.The Easy Drop Shape Analyzer (KRUSS GmbH) was used for contact angle measurement. Experimental results show that the wetting characteristics of the VACNT array surface can be modified from a contact angle of 140° to 0° depending on the distance between holes.

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