Generating Stable Cell Lines for HER2 and miR-7 Studies

MCF-7 cells were purchased from American Type Culture Collection and cultured according to their instructions. The stable MCF-7 cell line expressing pcDNA3 or the two independent cell lines expressing pcDNA3-HER2Δ16 and referred to here as MCF-7/pcDNA, MCF-7/HER2Δ16H, and MCF-7/HER2Δ16M1, respectively, have been described elsewhere [14] (link). For stable suppression of EGFR to generate the pooled MCF-7/HER2Δ16/EGFRKD cell line, MCF-7/HER2Δ16H cells were transfected with the MISSION shRNA plasmid-DNA TRCN0000121329 targeting EGFR (Sigma) or a pLKO.1 (Sigma) negative control using Fugene6 (Roche). For stable suppression of miR-7 to generate the pooled MCF-7/miR-7KD cell line, MCF-7 cells were transfected with the miRZip-7 anti-miR-7 microRNA construct MZIP7-PA-1 (System Biosciences) or the pGreenPuro Scramble Hairpin Control construct MZIP000-PA-1 (System Bioscience) using Fugene6. For stable expression of miR-7 to generate the pooled MCF-7/HER2Δ16H/miR-7 cell line, MCF-7/HER2Δ16H cells were transfected with the miR-7 expression vector MI0000263 (Origene) using the NEON Transfection System exactly as described by the manufacturer (Invitrogen). At two days post-transfection all pooled cell lines were selected for two days in 1 µg/ml puromycin (Gibco) and then maintained at 0.2 µg/ml puromycin.