Efficient transfection of primary zebrafish fibroblasts by nucleofection

Abstract

Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.

Fig. 1

Efficient transfection of zebrafish fibroblasts with pCS-GFP using nucleofection program O-20. (A–C) transfection of 2  ×  10⁶ primary cells ZEF1 resulted in 43% GFP-positive cells and 30% viability (A, B) as compared to the control cells that have been seeded without electroporation (C). (D–F) transfection of 4  ×  10⁶ primary cells ZEF2 resulted in 35% GFP-positive cells and 35% viability (D, E) as compared to the control cells (F). (G-I) transfection of 4  ×  10⁶ AB9 zebrafish fibroblast cell line resulted in 35% GFP-positive cells and 40% viability (G-H) as compared to control cells (I). Cells were seeded in 10 cm dishes. Images were taken 24-h after transfection using 10 ×  objective

Fig. 2

ZEF1 cells transfected with pH2B-GFP plasmid using nucleofection program O-20. (A and C) Nuclear localization of H2B-GFP in transfected cells. (B and D) Images of transfected cells in phase contrast. (A, B) zebrafish fibroblast cell line with a stable integration of pH2B-GFP. (C, D) H2B-GFP visualizes cell divisions. Images were taken one month after nucleofection and G418 selection. Scale bars correspond to 50 μm

Fig. 3

Differential transfection efficiency of zebrafish fibroblasts (ZEF1) using three optimized DNA delivery methods. The percentage of transfected cells was determined by cell counts 24-h post transfection. Data represent the mean ± standard deviation of four individual experiments. * p value < 0.05 relative to nucleofection values