e149 Nucleic Acids Research, 2007, Vol. 35, No. 22 Table 1. Oligonucleotide sequences and predicted transcripts Name FP-anta-18a RP-anta-18a anta-18a FP-anta-19b RP-anta19b anta-19b FP-anta-20a RP-anta-20a anta-20a FP-control RP-control anta-control impact on cell proliferation of miR-20a and miR-18a, respectively. MATERIALS AND METHODS Cloning of H1-antagomir expression cassettes Self-complementary DNA oligonucleotides (BioSpring, Frankfurt, Germany) encompassing the sequence of the miRNAs miR-18a, miR-19b and miR-20a, as well as an irrelevant sequence (ant-ctrl) were chemically synthesized including overhang sequences from a 50 Bgl II- and a 30 Sal I-restriction site. Annealed oligonucleotides were direc- tionally cloned into the Bgl II/Sal I-digested pBluescript- derived pH1-plasmid as described (14) to generate pH1ant-miR-18a, pH1ant-miR-19b, pH1ant-miR-20a and pH1-ant-ctrl). The oligonucleotide sequences are shown in Table 1. Each sense oligonucleotide harbors a stretch of T as a Pol III transcription termination signal, and the predicted transcript is indicated. pdc-SEW was used to generate lentiviral transgenic plasmids containing H1-antagomir expression cassettes located in the U3 region of the 30long-terminal repeat (LTR) (14). To generate the lentiviral pdcH1- antagomir-SEW plasmid, the pH1ant-mir-18a, pH1ant- mir-19b, pH1ant-mir-20a and pH1-ant-ctrl plasmids were digested with SmaI and HincII and the resulting DNA fragments ( 320 nt) were blunt-end ligated into the SnaBI site of pdc-SEW to generate pdcH1-ant-18a-SEW, pdcH1- ant-19b-SEW, pdcH1-ant-20a-SEW and pdcH1-ant-ctrl- SEW, respectively (Figure 1A). All lentiviral vectors encode EGFP as reporter gene driven by a viral SFFV (spleen focus forming virus)-LTR promoter and have an additional regulatory element of Woodchuck hepatitis virus (WPRE) for posttranscriptional processing. Lentiviral over-expression of miRNAs The retroviral vector MSCV/LTRmiR30-PIG RI con- taining the miR-30 backbone was kindly provided by Gerardo Ferbeyre (University of Montreal, Canada). To generate the lentiviral vector S-miR30miRNA-IEW, the plasmid pBluescript was digested with XhoI/HindIII PAGE 2 OF 9 Sequence (50–30) GATCCC CTATCTGCACTAGATGCACCTTA TTTTTTGGAAG TCGACTTCCAAAAAATAAGGTGCATCTAGTGCAGATAGG CUAUCUGCACUAGAUGCACCUUAUU GATCCC TCAGTTTTGCATGGATTTGCACA TTTTTTGGAAG TCGACTTCCAAAAAATGTGCAAATCCATGCAAAACTGAGG UCAGUUUUGCAUGGAUUUGCACAUU GATCCC CTACCTGCACTATAAGCACTTTA TTTTTTTGGAAG TCGACTTCCAAAAAATAAAGTGCTTATAGTGCAGGTAGGG CUACCUGCACUAUAAGCACUUUAUU GATCCC AAATCCTTTAGACCGAGCGTGTG TTTTTTTGGAAG TCGACTTCCAAAAAACACACGCTCGGTCTAAAGGATTTGG AAAUCCUUUAGACCGAGCGUGUGUU followed by a DNA polymerase I ﬁll-in reaction and gel puriﬁcation. The miR-30 cassette was excised from MSCV/LTRmiR30-PIG RI plasmid with AgeI/BglII, and the cohesive ends were ﬁlled in using Klenow. The blunt-end 390 nt fragment was ligated with the blunted XhoI/HindIII vector fragment. Self- complementary DNA oligonucleotides harboring the sequence of the miRNAs miR-18a, miR-19b and miR- 20a, were chemically synthesized including overhang sequences from a 50 XhoI- and a 30 EcoRI-restriction site for cloning purposes. Corresponding oligonucleotides were annealed and inserted into the XhoI/EcoRI-digested pBluescript plasmid pmiR30 to generate pmiR30miR-18a, pmiR30miR-19b, pmiR30miR-20a. The correct sequence and insertion was conﬁrmed by DNA sequencing for each plasmid. Finally, these plasmids were digested with SmaI and EcoO109I, and the resulting DNA fragments ( 500 nt) were blunt-end ligated into the BamHI site of the bicistronic plasmid SIEW harboring an IRES element (internal ribosome entry site) to generate S-miR30miR-18a-IEW, S-miR30miR-19b-IEW, S-miR30miR-20a-IEW and S-miR30-IEW (control), respectively (Figure 2A). All lentiviral constructs encode EGFP as reporter gene. Preparation of recombinant lentiviral supernatants and lentiviral transduction VSV.G-pseudo-typed lentiviral particles were generated by calcium phosphate co-transfection of 293T-cells (maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM), 10% FCS and 2 mM L-glutamine) and viral supernatants were concentrated as previously described (14). dcH1-antmiR-SEW lentiviral preparations were titrated in triplicate by serial dilutions of the concentrated vector stocks on 1 105 K562 cells (maintained RPMI 1640 with 10% FCS) in 24-well plates. The number of EGFP-positive cells were analyzed 72 h post-transduction by FACS analysis (FACS-Calibur, Becton-Dickinson, Heidelberg, Germany). The titers were averaged and typically ranged between 1–5 108 IU/ml. Lenti- and retroviral supernatants were used to trans- duce CML-derived K562 cells and to generate stable clones