Selected blood cell type-specific enhancer regions (ranging from 800-1200 bp) were PCR-amplified from human genomic DNA and cloned directly into the CpG-free pCpGL-CMV/EF1 vector 54,55 replacing the CMV enhancer with the DMR regions. Primer sequences are given in Supplementary Table 5. All inserts were verified by sequencing. For transient transfections, plasmids were isolated and purified using the EndoFree Plasmid Kit (Qiagen). Each luciferase construct was transiently transfected into three model cell lines (the monocytic THP-1 cell line, the Jurkat T cell line, and the B cell lymphoma cell line DAUDI). THP-1 and DAUDI cells were transfected using DEAE-dextran with 200 ng reporter plasmid and 10 ng Renilla control vector essentially as described56. Jurkat cells were transfected as described elsewhere54. The transfected cell lines were cultivated for 48 h, harvested, and cell lysates were assayed for firefly and Renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega) on a Lumat LB9501 (Berthold, Wildbach, Germany). Firefly luciferase activity of individual transfections was normalized against Renilla luciferase activity. Transfections correspond to at least three independent experiments measured in duplicates.
