Pgl3 promoter luciferase reporter plasmid

Firefly luciferase reporter vectors harboring the AR binding site of the MLPH gene, with the major T allele of SNP rs11891426:T>G, were constructed according to the procedures previously described (Bu, et al., 2013a (link)). Briefly, the MLPH ARBS was PCR amplified using primers F: 5′-TATCCAACACACGGGCTGAT -3′ and R: 5′-AGCTTTGGGGATTTCATTTCA -3′, purified and first ligated to the PCR fragment cloning vector PSC-B (Agilent Technologies, Santa Clara, CA) and then inserted into the KpnI/SacI sites of the PGL3 promoter luciferase reporter plasmid (Promega, Madison, WI). The minor G allele counterpart of the reporter vector or mutations of the two putative androgen-responsive elements (AREs) were generated using the QuikChange II site-directed (Agilent) or the Q5 site-directed mutagenesis (NEB, Ipswich, MA) kits. Primers used for mutagenesis were 5′-CAGCTCCCTGCTGCCAGCCTGGGG′ for G allele alteration, 5′-GCTTCCAGCCTGGGGTGCGTTCTGCACGCCTCCCTGAAATG for mutation of AR binding motif 3 and 5′-GCCAGCCCACAGCGTTCTGCACGCAGCCTGGGGTGGGAC for mutation of AR binding motif 2.