BOSC23 cells were maintained in high-glucose DMEM supplemented with 10% FBS and antibiotics. The following expression plasmids have been previously described: pHβAPNeo, pHβAP E47, pEBB RAG1 and pEBB RAG2 (8), pCSretTAC AS3 and pCSretTAC E47 (22), pRc/RSV CBP-hemagglutinin (HA) (23), and pCMVβ p300-CHA (24). BOSC23 cells were plated at 2 × 106 cells per 6-cm dish 1 d before transfection and transfected using Lipofectamine and Plus Reagent (Invitrogen).
BKO84 cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 5 × 10−7 M 2-mercaptoethanol and antibiotics (25). Retroviral vectors expressing a microRNA (miRNA)-based E2A knockdown construct or a negative control construct were generated as follows: oligonucleotides carrying an E2A target sequence or a LacZ control sequence were ligated into the pcDNA6.2-GW/miR vector (Invitrogen), and the resultant expression cassettes were transferred by Gateway Technology to the retroviral vector, MSCV GW PIG, which was generated by inserting the GW cassette into MSCV PIG dRI (26), kindly provided by Drs. S.W. Lowe and A. Yu (Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center [S.W.L.] and Cold Spring Harbor Laboratory [A.Y.]). Retroviral supernatants were prepared using the Plat-E packaging cell line (27), kindly provided by Dr. T. Kitamura (University of Tokyo). BKO84 cells were transduced with retroviruses and selected with 2 μg/ml puromycin after 2 d and used for assay at least after 7 d. Puromycin-resistant cells were cultured with 10 ng/ml PMA (Calbiochem) or vehicle control (DMSO) for 17 h (RT-PCR and chromatin immunoprecipitation assay) and 48 h (genomic PCR for rearrangement). Full paper
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