Limits...
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

Effect of DNA concentration on nucleofection frequency. GFP expression was analyzed by flow cytometry 48, 72, 96 and 168h after transfection of TO or ASK cells with varying amounts (2.5, 5 or 10 μg) of pmaxFP-Green-N. Data from a representative experiment (out of 3) is depicted here.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113957&req=5

Figure 2: Effect of DNA concentration on nucleofection frequency. GFP expression was analyzed by flow cytometry 48, 72, 96 and 168h after transfection of TO or ASK cells with varying amounts (2.5, 5 or 10 μg) of pmaxFP-Green-N. Data from a representative experiment (out of 3) is depicted here.

Mentions: The methods used are described in additional file 1. After testing the calcium phosphate transfection method and several commercially available lipid based formulations for transfection of salmon kidney cells without ever exceeding 10% efficiency, we decided to evaluate electroporation as an alternative. As our main long-term goal was to establish stably transfected cell lines from Atlantic salmon we first employed the pFRT plasmid containing Flp recombination target sequences that allow subsequent integration of the gene of interest using the Flp-In vector systems. To assess the potential transfection efficiency of salmonid cell lines, the pFRT-GFP-Zeo plasmid was transfected into TO cells by electroporation using a range of buffers and programs. Three different buffers in combination with eight different settings (electric pulse programs) were tested. This optimization kit contains enough material for one round of transfections. Directly after transfection, cell viability was assessed by trypan blue exclusion assay. Viability ranged from 72 to 100% with lowest mortality using buffer T (9%, not shown). Figure 1 and 2 (respectively) display cell viability (light diffraction) and EGFP expression (fluorescence) 3 days later. A combination of buffer T with pulse program 20 or 27 was optimal for both cell integrity and frequency of GFP expression (no significant difference). As pulse program T-20 gave higher viability directly after transfection, we used this program in subsequent experiments.


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)

Effect of DNA concentration on nucleofection frequency. GFP expression was analyzed by flow cytometry 48, 72, 96 and 168h after transfection of TO or ASK cells with varying amounts (2.5, 5 or 10 μg) of pmaxFP-Green-N. Data from a representative experiment (out of 3) is depicted here.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of DNA concentration on nucleofection frequency. GFP expression was analyzed by flow cytometry 48, 72, 96 and 168h after transfection of TO or ASK cells with varying amounts (2.5, 5 or 10 μg) of pmaxFP-Green-N. Data from a representative experiment (out of 3) is depicted here.
Mentions: The methods used are described in additional file 1. After testing the calcium phosphate transfection method and several commercially available lipid based formulations for transfection of salmon kidney cells without ever exceeding 10% efficiency, we decided to evaluate electroporation as an alternative. As our main long-term goal was to establish stably transfected cell lines from Atlantic salmon we first employed the pFRT plasmid containing Flp recombination target sequences that allow subsequent integration of the gene of interest using the Flp-In vector systems. To assess the potential transfection efficiency of salmonid cell lines, the pFRT-GFP-Zeo plasmid was transfected into TO cells by electroporation using a range of buffers and programs. Three different buffers in combination with eight different settings (electric pulse programs) were tested. This optimization kit contains enough material for one round of transfections. Directly after transfection, cell viability was assessed by trypan blue exclusion assay. Viability ranged from 72 to 100% with lowest mortality using buffer T (9%, not shown). Figure 1 and 2 (respectively) display cell viability (light diffraction) and EGFP expression (fluorescence) 3 days later. A combination of buffer T with pulse program 20 or 27 was optimal for both cell integrity and frequency of GFP expression (no significant difference). As pulse program T-20 gave higher viability directly after transfection, we used this program in subsequent experiments.

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