Figure 2.

Absence of RNAi in somatic cells. (A) A schematic composition of the Mos3 reporter transgene. (B) EGFP signal in different organs of MosIR, Mos3 and Mos3 & MosIR mice. Organs were collected from siblings of one litter. All images were taken with the same settings. The exposure of was shorter than in Figure 1C in order to obtain non-saturated brain EGFP signal. (C) qPCR analysis of different tissues shows little, if any, RNAi in somatic tissues and a strong RNAi effect in oocytes. Mos3 reporter expression in Mos3 & MosIR mice is shown relative to its expression in Mos3 mice. Samples were collected from two Mos3 and two Mos3 & MosIR animals and analyzed by qPCR in triplicates. Hprt was used as an internal normalization standard. Tissue heterogeneity could contribute to the lower level of Mos3 in the kidney. Error bars = SEM. (D) MosIR does not induce RNAi when transfected to somatic cells. The 293 cells stably transfected with a non-targeted firefly luciferase (FF) reporter and a targeted Renilla luciferase (RL) reporter (carrying Mos sequence in its 3′-UTR, Supplementary Figure S1B) were grown in 24-well plates and transfected with increasing amounts of the MosIR plasmid. pCAGEGFP was added to transfection mixtures to balance different amounts of the MosIR plasmid and to maintain the total amount of transfected DNA constant. Cells were harvested 48 h after transfection and luciferase activity was analyzed. Both luciferase activities are shown relative to cells transfected with 0 ng of the MosIR plasmid. NT = non-transfected cells. Transfection efficiency estimated by microscopic examination of EGFP fluorescence was >90%. Error bars = SEM.