Figure 2

Experiments with dicistronic vectors reveal a cryptic promoter. (a) The sequence under analysis was cloned in a dicistronic vector between two different luciferases from Renilla and firefly. HeLa and SH-SY-5Y cells were transfected with the indicated constructs. Cell lysates were prepared 24 hours post-transfection and the activity of the firefly luciferase was normalized to that of the Renilla luciferase. For each construct at least three independent experiments were performed. (b) The same sequence was cloned into a vector lacking the SV40 promoter (pRFΔP). Cell lysates were prepared 24 hours post-transfection and the activity of the firefly luciferase was normalized to that of the Renilla luciferase. For each construct at least three independent experiments were performed. (c) In vitro transcribed dicistronic mRNAs were synthesized from the indicated linearized constructs. HeLa cells were transfected with the capped dicistronic mRNAs and Renilla and firefly activities were measured 8 hours after transfection. Error bars represent standard error of the mean.