DNA methylation of the CMV promoter. a. The schematic diagram of the CMV promoter and the location of cytosine-phosphate-guanine dinucleotide (CpG) islands. After treatment with sodium bisulfite, genomic DNA was amplified with PCR primer JH1351 and JH1370. Red bar: CpG islands that were sequenced. b. DNA methylation of the CMV promoter using methylation-specific PCR (MSP). Stable clone cells that have the genomically integrated GBS-pCMV-copGFP were transiently transfected with synthetic suppressor vectors. Genomic DNA was extracted and amplified with primers that specifically recognize the methylated CpGs (top panel). Total genomic DNAs were amplified with primers that recognize both unmethylated and methylated CpGs. c. Efficiency of de novo DNA methylation by synthetic suppressors. Stable GBS-pCMV-copGFP clone cells were transiently transfected with 1 μg suppressor vectors. Forty-eight hours post-transfection, cells were harvested for bisulfate sequencing. DNA methylation was calculated as the average percentage of methylated CpGs/(methylated CpGs + unmethylated CpGs) from five CpG islands (110, 122, 141, 165, and 174). *P <0.05 as compared with cells transiently transfected with pcDNA3.1 empty vector.