a) TFBSs were predicted using Alibaba2 online software.
b) This sequence contains the human cytomegalovirus (CMV) immediate-early enhancer/promoter region and a chimeric intron.
Figure Legends
Figure 1. Effect of different promoters on transient transgene expression. pZsGreen1-1 vectors containing CMV, Actb, Ctsz, Hmox1, Hspa5, Rps18 and Vim promoters were transfected into CHO, HEK293T and BHK-21 cells. (A) ZsGreen1 fluorescence profile of transfected cells (100x, 200 ms exposure time). (B, D, F) %ZsGreen1 for CHO-K1, HEK293T and BHK-21 cells, respectively, co-transfected with new vectors carrying endogenous promoters and pBKS plasmid. (C, E, G) %ZsGreen1 for CHO-K1, HEK293T and BHK-21 cells, respectively, co-transfected with new vectors carrying endogenous promoters and pCIneo-CMV-IFNα plasmid. M2: positive cells with moderate fluorescence intensity, from 101 to 103 FAU (Fluorescence arbitrary units). M3:positive cells with high fluorescence intensity, FAU values higher than 103.
Figure 2. ZsGreen1 expression stability in CHO-K1 and BHK-21 cells lines grown until 45 and 50 generations, respectively, in the absence of neomycin selection pressure. Total %ZsGreen1 positive CHO-K1 and BHK-21 cells (A and B , respectively). Moderate ZsGreen1 MFI (M2) fold change in CHO-K1 and BHK1-21 cells (Cand E , respectively). High ZsGreen1 MFI (M3) fold change in CHO-K1 and BHK-21 cells (D and F , respectively).MFI: Mean Fluorescence Intensity. M2: positive cells with moderate fluorescence intensity, from 101 to 103 FAU (Fluorescence arbitrary units). M3:positive cells with high fluorescence intensity, FAU values higher than 103.
Figure 3. Impact of temperature shift on stable ZsGreen1 expression driven by endogenous promoters and CMV in CHO-K1 cells, during ten days upon cultivation at 37 °C or 32 °C. Bars show % of positive cells expressing moderate (black) and high (gray) levels of ZsGreen1 from samples taken on days 3, 5, 7 and 10. M2:positive cells with moderate fluorescence intensity, from 101 to 103 FAU (Fluorescence arbitrary units). M3: positive cells with high fluorescence intensity, FAU values higher than 103.