citations

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

Authors:

Schiøtz BL1, Rosado EG, Baekkevold ES, Lukacs M, Mjaaland S, Sindre H, Grimholt U, Gjøen T.

In:

Source: BMC Res Notes
Publication Date: (2011)
Issue: 4: 136

Research Area:

Cancer Research/Cell Biology

Gene Expression

Platform:

Nucleofector® I/II/2b

Experiment

Optimization with the Cell Line Optimization kit and the buffers, R, T and V. Test of the 7 Nucleofector 2 programs with clear identification of the best combination with Cell Line Kit T and T-020 program. In the fine tuning primary data show up to 90% of GFP positive cells after 48h. DNA titration shows effects from 1-2.5ug, more DNA does not have any positive effects on the transfection efficiency. All other chemical methods show below 10% transfection efficiency.

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.