Fig. 4

Percent of Transfected hMSCs Simultaneously Expressing Both Transgenes from Bi-Cistronic DNA Vectors. hMSCs were transfected with either the bi-cistronic pD2A or pIRES DNA vector complexed with Lipofectamine 3000 (a, c, e, & g) or Turbofect (b, d, f, & h) and assayed for transgene expression 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 pIRES. *** p < 0.001, **** p < 0.0001, as determined by a two-tailed T-test