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Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection.

Schiøtz BL, Rosado EG, Baekkevold ES, Lukacs M, Mjaaland S, Sindre H, Grimholt U, Gjøen T - BMC Res Notes (2011)

Bottom Line: Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry.A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells.The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway. tor.gjoen@farmasi.uio.no.

ABSTRACT

Background: Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines.

Findings: Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells.

Conclusions: The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

No MeSH data available.


Related in: MedlinePlus

Phase contrast and fluorescent micrographs (40X objective) of GFP positive TO cells after growth in normal medium (upper panels) and after constant selection with 0.05 mg/ml blasticidin (middle panels) or after selection with 0.05 mg/ml blasticidin for 2 weeks and then normal medium for 1 week (lower panels). Scale bar = 50 um.
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Figure 4: Phase contrast and fluorescent micrographs (40X objective) of GFP positive TO cells after growth in normal medium (upper panels) and after constant selection with 0.05 mg/ml blasticidin (middle panels) or after selection with 0.05 mg/ml blasticidin for 2 weeks and then normal medium for 1 week (lower panels). Scale bar = 50 um.

Mentions: Blasticidin resistant and GFP-expressing cells were obtained by transfection with the pSELECT-blasti-mcs plasmid followed by selection. In cultures not selected with blasticidin, the culture was dominated by cells not expressing GFP (Figure 4, upper panel). In cultures selected for 3 weeks with 0.05 mg/ml blasticidin, many cells were GFP positive, but poor growth was observed (Figure 4, middle panel). However, in cultures where selective medium was replaced with normal medium for one week, growth was restored and the fraction of GFP positive cells was further increased (Figure 4, lower panel). When cells were selected with 0.01 mg/ml blasticidin, a lower fraction of GFP positive cells were observed (not shown).


Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection.

Schiøtz BL, Rosado EG, Baekkevold ES, Lukacs M, Mjaaland S, Sindre H, Grimholt U, Gjøen T - BMC Res Notes (2011)

Phase contrast and fluorescent micrographs (40X objective) of GFP positive TO cells after growth in normal medium (upper panels) and after constant selection with 0.05 mg/ml blasticidin (middle panels) or after selection with 0.05 mg/ml blasticidin for 2 weeks and then normal medium for 1 week (lower panels). Scale bar = 50 um.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113957&req=5

Figure 4: Phase contrast and fluorescent micrographs (40X objective) of GFP positive TO cells after growth in normal medium (upper panels) and after constant selection with 0.05 mg/ml blasticidin (middle panels) or after selection with 0.05 mg/ml blasticidin for 2 weeks and then normal medium for 1 week (lower panels). Scale bar = 50 um.
Mentions: Blasticidin resistant and GFP-expressing cells were obtained by transfection with the pSELECT-blasti-mcs plasmid followed by selection. In cultures not selected with blasticidin, the culture was dominated by cells not expressing GFP (Figure 4, upper panel). In cultures selected for 3 weeks with 0.05 mg/ml blasticidin, many cells were GFP positive, but poor growth was observed (Figure 4, middle panel). However, in cultures where selective medium was replaced with normal medium for one week, growth was restored and the fraction of GFP positive cells was further increased (Figure 4, lower panel). When cells were selected with 0.01 mg/ml blasticidin, a lower fraction of GFP positive cells were observed (not shown).

Bottom Line: Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry.A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells.The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway. tor.gjoen@farmasi.uio.no.

ABSTRACT

Background: Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines.

Findings: Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells.

Conclusions: The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

No MeSH data available.


Related in: MedlinePlus