Pcpgfree promoter

To study the regulatory consequence of in vitro methylation of the targeted CpG island, we first used the commercially available reporter plasmid pCpGfree-promoter as the backbone (Invivogen, San Diego, CA). This plasmid containing both a sequence for secreted luciferase and a promoter devoid of CpG dinucleotides, which renders it insensitive to DNA methylation. We then synthetized the Gpx1 CpG island region by PCR amplification using forward 5’-TATAAGCTTGAAGTGAATGGTGAGAAGGCTCACCCGC-3’ and reverse 5’- ATATGTACAACCAAGCCAATGCCAGGGCCGCCTTAG-3’ primers on mouse genomic DNA as a template. This newly generated CpG-rich sequence was then inserted in the pCpG free-promoter plasmid using HindIII and BsrGI restriction sites. We named the resulting vector “pCpG free-Gpx1” (see “results”).

Constructions were done using the CpG free plasmid pCpGfree-promoter (Invivogen, San Diego, CA) as the backbone to study enhancer methylation. The ANGPTL2 CpG region was amplified using forward 5’-TAAGCTCCTTCCCACGTGACCTCACAGAGTCG-3’ and reverse 5’-GATCCGACTCTGTGAGGTCACGTGGGAAGGAGCTTATGCA-3’ primers and subsequently inserted in the backbone using NsiI and BamHI restriction sites as previously described [54 , 55 (link)].