Generating Hybrid Promoter Constructs for Gene Expression Analysis

The pDRAprox vector has been previously described (Krawczyk et al., 2007 (link)). This vector contains an HLA-DRA promoter fragment (from −151 to +10) inserted upstream of the firefly luciferase reporter gene in the pGL3b vector (Promega). The vector containing the BTN2A2 promoter was generated by inserting a PCR-amplified fragment encompassing the SXY module and transcription start site of BTN2A2 between the MluI and BglII sites of pGL3b. Vectors containing hybrid promoters were generated by replacing the HLA-DRA SXY module with BTN SXY fragments using the MluI–BglII sites in pDRAprox. Mutations were introduced as previously described (Krawczyk et al., 2007 (link)). Primers used for cloning are available upon request. Raji, RJ2.2.5, and SJO cells were cotransfected with the firefly luciferase reporter vectors and a control renilla luciferase vector. Transfections were performed by electroporation (950 µF and 0.21 V). Dual luciferase assays were performed as previously described (Krawczyk et al., 2007 (link)).