To prepare and transfect (and see ref. 17) the unmethylated and methylated plasmids, we subjected the exon 1F NR3C1 promoter to PCR amplification and cloned the resulting PCR product into a PCR2.1 plasmid (Original TA cloning kit, Invitrogen). The NR3C1 promoter was then ligated into the PCR2.1 plasmid. For patch methylation, the glucocorticoid receptor 1F plasmid was incubated (2 h, 37 °C) with SssI DNA methyltransferase (20 U, New England Biolabs) in a buffer containing S-adenosylmethionine, and this procedure was repeated until full protection from HpaII digestion was observed. Following digestion with HindIII and EcoRV restriction enzymes, each fragment was then ligated into a pGEM-luc vector (Promega) at the HindIII and BamHI or XbaI and BamHI sites in the 5′ to 3′ (sense) or 3′ to 5′ (antisense) orientation, respectively. The concentration of each ligation product was determined by fractionation on a 1.5% agarose gel, by comparing bands of the expected ligation product size against a standard curve of known DNA concentration (ten fivefold serial dilutions of 2 µg/µl−1 micrococal nuclease DNA) to control for possible unequal efficiency of ligation
