Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12 °C

Marivin, Elisa; Mourot, Brigitte; Loyer, Pascal; Rime, Hélène; Bobe, Julien; Fostier, Alexis

doi:10.1016/j.ygcen.2015.03.012

Cited by 3 publications

(3 citation statements)

References 33 publications

(26 reference statements)

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“…This is the first report of electroporation of protein: RNA complex in salmonid cells. The differences in optimal electroporation settings compared with previous studies using plasmide may be explained by the difference in cargo or the use of OptiMEM instead of buffer R for the electroporation (Ojima et al 1999 ; Chi et al 2012 ; Marivin et al 2015 ). These settings were also optimal for electroporation and editing using Cas12a RNP in both Atlantic salmon and rainbow trout cell lines.…”

Section: Discussionmentioning

confidence: 98%

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

et al. 2020

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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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Section: Discussionmentioning

confidence: 98%

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

et al. 2020

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“…The obtained high transfection is remarkable given that transfection is key to high gene editing efficiency, and salmonids cells-including ASK-1, SHK-1 and CHSE-214-are intrinsically difficult to transfect [28] due to the low incubation temperature (15-22 • C) and high saturation of the phospholipids in the cellular membrane compared to mammalian cells [29]. The comparative low transfection efficiency (1-5%) by the plasmids (px458 and px459) can be attributed to a difference in cargo, because the low transfection efficiency of salmonids cells by plasmids (both by electroporation and chemical mediated transfection) has generally been reported [30,31]. Two studies [30,31] previously reported up to 40% positive plasmid electroporation, although the plasmids used in these studies were not adapted for CRISPR/Cas gene editing in contrast to px458 and px459, which, in addition to sgRNA, also encoded the GFP and puromycin markers, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The comparative low transfection efficiency (1-5%) by the plasmids (px458 and px459) can be attributed to a difference in cargo, because the low transfection efficiency of salmonids cells by plasmids (both by electroporation and chemical mediated transfection) has generally been reported [30,31]. Two studies [30,31] previously reported up to 40% positive plasmid electroporation, although the plasmids used in these studies were not adapted for CRISPR/Cas gene editing in contrast to px458 and px459, which, in addition to sgRNA, also encoded the GFP and puromycin markers, respectively. Transfection is, thus, an important parameter to be optimized in the use of the plasmid-mediated CRISPR/Cas strategy for the gene editing of fish cell lines that grow at low temperatures.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

Strømsnes

Schmidke

Azad

et al. 2022

IJMS

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Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci—cr2 and mmp9 of A. salmon—were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells.

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Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

Gratacap

Jin

Mantsopoulou

et al. 2020

Preprint

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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 a valuable model to study infectious diseases in salmonids, and genome editing using CRISPR provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas9 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.

show abstract

Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12 °C

Cited by 3 publications

References 33 publications

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes

CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

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