PMC:3320587 / 11230-12646 JSONTXT

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Lentiviruses encoding shRNA sequences were generated by transfecting the packaging cell line HEK-293T with the shRNA-encoding pLKO.1 plasmids in combination with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Effectene transfection reagent (Qiagen, www.qiagen.com). Supernatants were collected 48 hours post-transfection, clarified by centrifugation, and stored at −80°C. THP-1 cells were transduced with the lentiviral particles by culturing the cells with supernatants from the virus-producing cells in the presence of 8 µg/ml polybrene (Millipore, www.millipore.com) and spinoculation for two hours at 2000 RPM. 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