Figure 4

TRBP is required for RNA interference mediated by shRNA or exogenous miRNA against EGFP in murine cells. A) Schematic representation of the sh and miRNAs transcribed from pCRII-U6+27 vectors and targeting non specific (ns) or EGFP mRNA. The guide strand is shown in red. The seed sequence is highlighted in blue. B, C, D) sh and miRNA against EGFP in murine cells are active only when TRBP is present. MEFs (left panels), TEFs heterozygous for TRBP expression (tarbp2^+/-; middle panels) or TEFs disrupted for TRBP expression (tarbp2^-/-; right panels) were transfected with 1 μg of EGFP-C1 and 4 μg of ns or anti-EGFP sh- or mi-RNAs, as indicated. B) Fluorescence of live cells was monitored on an inverted microscope and a representative picture is shown. Percentages reflect knock-down of EGFP expression as calculated by mean luminosity intensity. C) Cells were analyzed by fluorescence automated cell sorting (FACS). Reporter expression is calculated as the percentage of EGFP expression in the presence of sh- or miRNA compared to ns normalized to 100%. Values are means of four independent experiments ± SEM. D) Total cell extracts were analyzed by immunoblot for EGFP and actin expression. Percent decrease of EGFP signal was calculated by densitometry analysis.