Hi,
I would like to generate a cell line stably overexpressing a gene of interest. We normally use a system containing a CMV promoter. However I wonder if methylation of the promoter could occur along passages and finally silence my gene. Would it be better to use a CpG-free promoter or other type of sequence to drive the expression? if anyone has already tested those construct, thanks for your feedbacks.
The CMV promoter is considered to be a strong promoter; however it is subject to silencing. I am unaware of any CpG-free promoter sequences and would like to hear answers from others if they exist and if they are strong promoters. To overcome silencing people have invested the use of MAR and UCOE elements.
Thanks Paula
I found a CpG free promoter sold by Invivogen. I think it is derived from the plasmid published in "CpG-Depleted Plasmid DNA Vectors with Enhanced Safety and Long-Term Gene Expression in Vivo" and it is basically used for methylation assays. Do you have a plasmid you would recommend having the MAR and UCOE elements?
Dear Chaveroux
The plasmid pRB7-6 (Plasmid #24700) containing Tobacco Rb7 MAR (SAR).
https://www.addgene.org/24700/.
Best,
thank you David, that was exactly what i was looking for. I will read in deep the original paper and probably ask for the plasmid.
last question David, have you ever tried to use the construct containing the 2 insulators and the chicken promoter in mouse or human cell lines?
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