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.

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 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.

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 A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marina Zoppo ◽  
Nicole Okoniewski ◽  
Stanislav Pantelyushin ◽  
Johannes vom Berg ◽  
Kristin Schirmer
Keyword(s):  
Rainbow Trout ◽  
Single Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Gene Editing ◽  
Cell Clone ◽  
Intestinal Cell ◽  
Fish Cell ◽  
Sequencing Analysis ◽  
Mammalian Cell Lines

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.

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 Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 690
Author(s):  
Ke Zhang ◽  
Wenzhi Liu ◽  
Yiqun Li ◽  
Yong Zhou ◽  
Yan Meng ◽  
...  
Keyword(s):  
Cell Lines ◽  
Grass Carp ◽  
Amino Acid Sequences ◽  
Gibel Carp ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
Total Size ◽  
Sequence Comparisons ◽  
Grass Carp Reovirus

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/μL.

Development, characterization, conservation and storage of fish cell lines: a review

2010 ◽  
Vol 37 (1) ◽  
pp. 1-20 ◽  
Author(s):  
W. S. Lakra ◽  
T. Raja Swaminathan ◽  
K. P. Joy
Keyword(s):  
Cell Lines ◽  
Fish Cell ◽  
Fish Cell Lines ◽  
And Storage
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