help, methylated CMV promoter inactivated - (Jul/04/2006 )

Hi, all.

We want to eliminate unmethylated CpG elements to reduce inflammatory activity of plamsid DNA. We have seen >10 fold reduction in promoter (CMV) activities after SssI methylase methylation compared to untreated plasmid. Can experts here provide me with an explanation what causes this inactivation? Is that because new proteins bind specifically to the methylated DNA that led to the inactivated transcription? How do I reduce such effect? Any suggestion is greatly appreciated.

We know that promoter methylation can silence gene transcription via multiple mechanisms such as methylated CpGs attract methy-cpg binding proteins or interfere directly with transcriptional factor binding. For a particular promoter, it's hard to tell which mechanism is responsible. In your situation, you need a method to protect the CMV promoter from methylation during methylase treatment. This protection thus is sequence-specific. How about targeting the promoter with an oligo which is homologus to the promtoer so that a triple helix can form at the promoter. Will the triple helix prevent methylation???

Hi Genehunter, did the triplex method work? If it's good I'll try it too...