Fig 1.
Schematic of DNA vectors tested.
Vectors are drawn to relative scale. p = plasmid; mv = minivector. The numbers denote length in base pairs.
Fig 2.
Effect of DNA vector length on cell entry.
Each of the DNA vectors was labeled with Cy3 using LabelIT and transfected into 90,000 HeLa-GFP cells. Cells were transfected using electroporation with 30 ng of each of the eight DNA vectors and the median Cy3 fluorescence was determined using flow cytometry (circles). In a separate experiment, 90,000 HeLa-GFP cells were transfected with 17 fmol of each of the eight DNA vectors (equivalent to 30 ng of the p2844) (squares). Median Cy3 fluorescence was compared to a standard curve of R-PE beads to determine the number of fluorophores, which, when combined with the number of Cy3 fluorophores per DNA molecule for each vector, yielded the number of molecules per cell. *For transfections at equal mass, results for mv727 were statistically significantly higher (by one-way ANOVA) than the results for all the vectors except mv1018. No other pairwise comparisons were statistically significant.
Fig 3.
Lack of correlation between labeled DNA cell entry and GFP knockdown.
HeLa-GFP cells were electroporated with Cy3-labeled DNA vectors. Representative results for the mv383 and p2844 vectors are shown. (A) GFP versus Cy3 fluorescence for cells transfected with 30 ng of mv383 or p2844 (B) Gating of GFP positive (green) and GFP negative cells (grey) (C) Cy3 fluorescence of GFP positive (green) versus GFP negative cells (grey) as gated in (B).
Fig 4.
Flow cytometric analysis of the effect of DNA vector length on GFP knockdown.
(A) Fluorescence of positive and negative controls showing the threshold between “GFP positive” and “GFP negative” cells. (B) 90,000 HeLa-GFP cells were transfected using electroporation with 50 ng of each of the eight DNA vectors and analyzed using flow cytometry (circles). In a separate experiment, 90,000 HeLa-GFP cells were transfected with 17 fmol of each of the eight DNA vectors (equivalent to 50 ng of the p4548) (squares). (C) p-values for a one-way ANOVA for pairwise comparisons of all the vectors are given, and statistically significant differences are highlighted.
Fig 5.
Fluorescence microscopy analysis of the effect of DNA vector length on GFP knockdown.
90,000 HeLa-GFP cells were transfected with the indicated concentrations of each vector, transferred after 24 hours to a 96-well glass bottom plate, incubated for another 24 hours, then fixed. Photographs of fields of cells from several wells from each transfection were taken. Images were analyzed using myImageAnalysis [21] to quantify GFP fluorescence and size for each cell, encompassing several thousand cells in each transfection. Data were plotted using a Tukey box plot where each outlier cell, defined by having a GFP per cell area more than 1.5 x interquartile range above the 75th percentile, is denoted by a circle. Statistical differences were determined using a one-way ANOVA on ranks, post hoc Kruskal-Wallis test. In the top panel, all results except for those from mv383 and mv727 were statistically different from each other. In the middle panel, all but the two control samples were statistically significantly different from each other. In the bottom panel, all transfected cells were statistically significantly different from the controls, but only results for cells transfected with mv1018 differed from the other vectors.
Fig 6.
Direct comparison of transfection efficiency for three pairs of vectors.
The larger vectors (dashed dark blue lines) were each transfected at one of three concentrations (mass and moles for these vectors are given) into 90,000 HeLa-GFP cells. For each concentration of the larger vector, each smaller vector was separately transfected into HeLa-GFP cells at two different concentrations. One concentration of the smaller vector was equivalent to an equal mass of minivector (solid light blue line), and the other concentration was equivalent to an equal number of molecules of smaller vector (dotted light blue line). Cells were analyzed using flow cytometry 48 hours post-transfection. Each panel is a representative result of an experiment that was performed at least twice in duplicate.
Fig 7.
Effect of GFP knockdown as a function of DNA concentration.
GFP knockdown was measured using a variety of DNA concentrations for each of the eight DNA vectors. Cells were analyzed using flow cytometry 48 hours post-transfection. The data were plotted either comparing the mass (A) or the number of molecules (B) of each of the different DNA vector lengths. Each panel shows the percentage of cells where GFP has been knocked down relative to the “no DNA” controls.
Table 1.
Two-way ANOVA showing statistically significant effects of vector size.
P-values of pairwise comparison of vectors after taking mass (top) and moles (left) into account.