Figure 2.

Monitoring transfection of rainbow trout red blood cells (RBCs). Fluorescent micrographs of RBCs transfected with (A) pmTFP1 and (B) pmTFP1GVHSV at six days post-transfection at 14°C, monitored by teal fluorescent protein (TFP). Fluorescent images taken with the IN Cell 6000 imaging system, augmentation 60x. (C) RBC transfection with 2, 4, and 8 μg of plasmid pmTFP1GVHSV was confirmed by GVHSV gene RT-qPCR, at one day (white bars), three days (gray bars) and six days (black bars) post-transfection. The eukaryotic 18S rRNA gene was used as an endogenous control. Data are displayed as mean ± SD (n = 3). Two-way ANOVA with Tukey's multiple comparisons test was performed between plasmid concentrations (black lines and asterisks) and times post-transfection (gray lines and asterisks). (D) Time course of transfected RBCs (black bars) and transfected RTS11 (gray bars) with 4 μg of pmTFP1GVHSV at one, three and six days post-transfection monitored by GVHSV RT-qPCR. The eukaryotic 18S rRNA gene was used as an endogenous control. Data are displayed as mean ± SD (n = 3 for RBCs and n = 2 for RTS11). Two-way ANOVA with Sidak's multiple comparisons test was performed between cell types at the different times post-transfection. ^*, ^**, ^***, and ^****, represent the P values < 0.05, < 0.01, < 0.001, and < 0.0001, respectively. Overlay flow cytometry histogram of representative GVHSV immunostaining of (E) RBCs and (F) RTS11 cells transfected with 4 μg of pmTFP1 or pmTFP1GVHSV at three days post-transfection (gray histogram, pmTFP1-transfected; green histogram, pmTFP1GVHSV-transfected). Representative immunofluorescence of GVHSV intracellular immunostaining of (G) RBCs and (H) RTS11 cells transfected with 4 μg of pmTFP1 or pmTFP1GVHSV at three days post-transfection (protein [APC, red] and nuclei [DAPI, blue]). Fluorescence images were taken with 60 × magnification.