Fig. 6

Comparison of Percent of Transfected hMSCs Simultaneously Expressing Both Transgenes from Delivery of Two DNA Vectors to Delivery of Bi-Cistronic DNA Vectors when Equal Mass of DNA and Copy Number of Transgenes are Delivered.  hMSCs were transfected with single transgene DNA vectors delivered in separate complexes ([pE]+[pT]), the same complex ([pE + pT]) or bi-cistronic DNA vectors (pD2A or pIRES) with an equal mass of DNA and copy number of transgenes delivered. All four conditions were complexed with Lipofectamine 3000 (a, c, e, & g) or Turbofect (b, d, f, & h). Transgene expression for each condition was assayed 24 h after transfection for D1 hAMSCs (a & b), D2 hAMSCs (c & d), D3 hBMSCs (e & f), and D4 hBMSCs (g & h). The percent of EGFP expressing cells that are expressing tdTomato was calculated by dividing the number of EGFP positive cells that were also tdTomato positive by the number of EGFP positive cells. The reasoning behind normalizing to EGFP positive cells, as well as counting EGFP positive cells that are also tdTomato positive, as opposed to normalizing to tdTomato positive cells or tdTomato positive cells that are also EGFP positive, is that translation of the upstream transgene (EGFP) is required for D2A sequences before translation of the downstream transgene can occur. Data represented as mean ± SEM (n = 6). * indicates significance relative to [pE]+[pT]. *** p < 0.001, **** p < 0.0001. # indicates significance relative to [pE+pT]. # p < 0.05, ## p < 0.01. $ indicates significance relative to pIRES. $ $ p < 0.01, $ $ $ p < 0.001, $ $ $ $ p < 0.0001, as determined by a 2-way ANOVA with Tukey’s post hoc test. Only significant comparisons are shown for clarity