Luciferase reporter plasmids for miRNA regulation

The luciferase reporter plasmids PGK-FL-let-7-3xP-BGHpA, PGK-FL-let-7-4xB-BGHpA, and PGK-FL-miR-30-4xB-BGHpA used to produce reporter cell lines for HTS were built stepwise on the HindIII-AflII pEGFP-N2 (Clontech) backbone fragment using PCR-amplified fragments carrying appropriate restriction sites at their termini. First, we produced a basic firefly luciferase reporter composed of PCR-amplified fragments. The PGK promoter sequence was amplified from the phRL_PGK plasmid (Ma et al., 2010 (link)), the firefly luciferase coding sequence was obtained from the pGL4.10 plasmid (Promega), the BGHpA sequence from the pcDNA3.1(–) plasmid (Invitrogen), and a synthetic polyA signal (SpA) sequence upstream of the PGK promoter was taken from the pGL4.10 plasmid. PCR primers used in the cloning are listed in Table S1. Finally, the miRNA binding sites were inserted into the plasmid using in vitro synthesized oligonucleotides carrying miRNA binding sites for let-7 or miR-30 miRNA, which were annealed and cloned into a BamHI site downstream of the luciferase CDS; the plasmids were validated by sequencing. The pGL4_SV40_1xmiR-30P plasmid was generated by inserting the fragment with the miR-30 1xP binding site from phRL_SV40_1xmiR-30P (Ma et al., 2010 (link)) into pGL4_SV40 using XbaI and ApoI restriction sites.