Figure 6.

Coordinating endosomal escape and intracellular trafficking for efficient ex vivo modification. (A) Neural stem cells (16) were transfected with Transficient/pmaxGFP (MBL International Corp./Lonza) at various amount of DNA. Cells were transfected following manufacturer's instruction or centrifugation procedure. Following centrifugation, cells were treated with DOPE/CHEMS and 10 μM Tubastatin A. Data presented as mean ± SD (n = 3). For cells transfected at >250 ng of pDNA (manufacturer's instruction), transfection efficiencies were not analyzed due to the low remaining cell number. (B) Human dermal fibroblast (Cell Applications) was transfected with Transficient/500 ng pmaxGFP using centrifugation procedure, with or without DOPE/CHEMS plus 10 μM Tubastatin A. Data presented as mean ± SD (n = 3). Representative images are presented. Significant differences in transfection efficiencies were calculated using two tailed Student's t-test. **P < 0.005. (C) Complexes of Transficient/EGFP_mRNA (Trilink), at various amount, were prepared. MSC were transfected using manufacturer protocol or centrifugation method. After centrifugation, cells were treated with DOPE/CHEMS plus Tubastatin A (10 μM). Data presented as mean of biological duplicates. Representative images are presented. In all cases, transfection efficiency was acquired with FACS, 24 h post-transfection. Transfection efficiency was calculated as the percentage of EGFP+ cells normalized to the total number of cells as quantified by FACS.