Pcpg free basic lucia

For in vitro methylation studies, constructions were done using the CpG-free and promoterless vector pCpG-free basic-Lucia (Invivogen, San Diego, CA) as the backbone. A 469-bp Sod2 promoter region was amplified on mouse genomic DNA using forward BamHI (5′-ATTGGATCCTTTGCAGCTCACAGCCAGAGCTGGACA-3′) and reverse HindIII (5′-TAAAAGCTTAGCCAGCCACGCCCGCCGCCCCG-3′)-linked primers and subsequently inserted in the backbone using BamHI and HindIII restriction sites. Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). M.SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer's instructions. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop Products, Wilmington, DE). Methylation was confirmed by digestion with the methylation-sensitive restriction enzymes HhaI and HpaII. HEK293 cells grown to confluence on 96-well plates were transfected with the pCpG-free Sod2 vector using Lipofectamine 2000 (Invitrogen). Following 24 h transfection, luciferase activity was measured with the QUANTI-Luc reagent (Invivogen) by luminescence detection.

The PCR fragments of −828/−1 of human CAMP were amplified from human blood genomic DNA (Promega, Madison, WI) using primer pairs P1/P2 (Table 3) and Herculase^® II Fusion DNA Polymerase (Agilent technologies, Santa Clara, CA). Amplified fragments were inserted into the pGEM-T vector (Promega, Madison, WI) to generate plasmid pGEM-T-828, which was verified by DNA sequencing. A 845 bp fragment amplified with primer pair P3/P4 (Table 3) was ligated into a Lucia reporter plasmid pCpGfree-basic-Lucia (Invivogen, San Diego, CA) without a promoter and devoid of CpG dinucleotides via the AvrII and BamHI restriction sites to generate vector pCpGfree-CAMP (−828/−1). Constructs were confirmed by automated sequencing (Sangon, Shanghai, China).