Figure 2

Effect of the 5′ mRNA cap structure on expression of the encoded protein in DCs. (a, b) Human immature and mature DCs (iDCs and mDCs, respectively) were electroporated with equal amounts of RNAs encoding reporter genes (luciferase or d2eGFP) transcribed in the presence of the indicated cap analogs or capped post-transcriptionally using the capping enzyme from vaccinia virus (m⁷GpppG/p.t.). ApppG with adenosine instead of guanosine, and GpppG, which lacks the methyl-group essential for recruitment of the translational machinery, were used as non-functional cap controls. (a) Luciferase-encoding RNAs were used as reporters, luciferase activity (given in counts per second, cps) was measured, and the average values±s.d. are shown as a function of time. On the basis of the data shown here the translational efficiency, the time-point of the maximal protein expression, and the total protein expression as given in Table 1 were calculated. (b) d2eGFP-encoding RNAs were used as reporters, and fluorescence given in mean fluorescence intensity was measured by flow cytometry. For unknown reasons the expression from m7GpppG/p.t. RNA was unusually low in immature DCs in the particular experiment shown here.