PAGE 5 OF 9 Nucleic Acids Research, 2007, Vol. 35, No. 22 e149 Figure 2. Lentivirus-mediated miRNA-expression. (A) Schematic representation of the lentiviral transgene plasmid harboring a miRNA-expression cassette. Speciﬁc miRNA sequences are embedded within sequences derived from miR-30 as described (17). S:SFFV-LTR, I: IRES, E:EGFP, W:WPRE. (B) miRNA expression in control shRNA expressing K562 cells upon lentiviral transduction with miR-18a, miR-19b and miR-20a as determined by miR-qRT-PCR. Mean of two independent experiments are shown. (C) Luciferase activity in K562 cells with reduced c-MYC expression stably transfected with miR-18a sensor (left), miR-19b sensor (middle) and miR-20a sensor (right) luciferase constructs upon lentiviral expression of corresponding control, miR-18a, miR-19b and miR-20a miRNAs, respectively. The ratio of normalized sensor to control luciferase activity is shown as in Figure 1B. Mean of two independent experiments are shown. (D) Proliferation of K562 cells expressing control (left) and anti- c-myc shRNAs (right) upon lentiviral transduction for miRNA expression as indicated. Note the diﬀerent y-axes in both panels due to impaired cell growth of K562 cells with reduced c-MYC expression (7). (E) Cell proliferation of K562 cells expressing diﬀerent miRNAs in the presence of lentivirally encoded control antagomirs (upper graph), anti-miR-18a antagomirs (middle graph) and anti-miR-20a antagomirs (lower graph), respectively. Antagomir-transfected K562 cells were transduced with miR-30 control (control), the miR-17-19b cluster (miR17/19b), miR-18a (miR-30-18a), or miR-20a (miR-30-20a), and the number of trypan blue negative cells is indicated as described for Figure 1F–H. Mean of two independent experiments are shown. used for visualization using the ECL western blotting detection reagents (Amersham Biosciences) according to the manufacturer. Densitometry was done on scanned images using Adobe Photoshop 7.0 software. RESULTS Generation of antagomir-encoding lentiviruses To generate stable miRNA loss-of-function phenotypes, we used our previously described lentiviral vectors, which encode shRNAs and allow stable transduction and isolation of transduced cells based on lentivirus-mediated EGFP expression (14). In this system, ﬂuorescence intensity of the reporter gene product correlates to the level of shRNA expression and, correspondingly, to the amount of RNAi-mediated inhibition of gene expression. Accordingly, we cloned antagomir-encoding oligonucleo- tides downstream of a H1-promoter located in the U3 region of the 30LTR (Figure 1A). During reverse transcription, the H1-promoter-antagomir expression cassette is transferred to the 50LTR resulting in two