Transiently transfected luciferase reporters are inhibited by expressed dsRNA.

<p>(A) The inhibition of reporter activity is independent of the hairpin RNA sequence and it is absent when the inverted repeat is placed downstream of non-active ZP3 promoter. HEK-293 cells were transiently transfected with 100 ng/well of each RL (triangle) and FL (square) reporter plasmids and increasing amount (0–250 ng/well) of either pCAGEGFP-Lin28IR (containing an active promoter) or pZP3EGFP-Lin28IR (containing an inactive promoter). Luciferase activity was analyzed 48 hours after transfection. pBluescript was added to maintain the amount of transfected DNA constant. Both luciferase activities are shown relative to cells transfected with 0 ng of the hairpin-expressing plasmid. (B) Similar to (A) except Elavl2IR-expressing plasmids (pCAGEGFP- Elavl2IR or pZP3EGFP-Elavl2IR) were used. Data are shown as an average of at least 3 experiments made in triplicates. Error bars = SEM. (C, D) A hairpin-expressing pCAGEGFP-MosIR plasmid affects the luciferase activity of transiently transfected but not stably integrated reporter plasmids. HEK-293 cells with stably integrated RL and FL reporters (C), or HEK-293 cells with stably integrated RL reporter only (D) were transiently transfected with an increasing amount of pCAGEGFP-MosIR and a constant amount of FL reporter (if not stably integrated). pBluescript was added to maintain the amount of transfected DNA constant. Both FL (squares) and RL (triangles) luciferase activities were analyzed 48 hours post-transfection. Luciferase activities in cells transfected with 0 ng of the pCAGEGFP-MosIR were set to one. Data show an average of at least 3 experiments done in triplicates. Error bars = SEM.