I am trying to understand if the promoter that I want to stably transfect into the cells through the use of a plasmid could be epigenetically modified, as occurred for the endogenous promoter.
Thanks.
Per Tanti's remark: Introduction of NeoR and the use of any aminoglycoside (G418, Neomycin, Kanamycin, Streptomycin, etc.) will lead to the production of an inhibitor of ATP-dependent chromatin remodeling thereby disrupting "normal" epigenetic processes in eukaryotes. Epigenetic compensation by other non-ATP-dependent systems is likely required for cell survival. This will also happen with empty vector NeoR controls and hence the dearth of those controls in the literature. Bottom line: If you are using NeoR in eukaryotes and cannot establish an empty vector control that is pheotypically identical to the parent cell, you should assume that there is epigentic compensation and seriously consider switching to puroR or some other marker.
That is the way Rob Waterland demonstrated that epigenetics can be modified during pregnancy when fetus is exposed to specific molecules.
Article Transposable Elements: Targets for Early Nutritional Effects...
Yes, transfected plasmids can get chromatinized and consequently could gain DNA methylation. The status of CpG methylation of your original DNA is also important.
You could start here:
http://www.sciencedirect.com/science/article/pii/0014579394003947
Good luck.
Antoine.
As mentioned above, this could happen quite frequently. Indeed, it is possible to have a stable cell line with a transgene whose expression could be down-modulated over time. I also had problems to keep transgene expression stable over time, although the cell line was under antibiotics selection. The simplest way to avoid it, in my opinion, is to keep cells in culture for the shortest possible time.
epigenetic modifications are inevitable but you can have your own and defined stable condition which in comparison with your control samples you can judge your results.
Good luck
Well we have also seen this type of epigenetic modification in our system that is the epigenetic modification and its effect in transgene expression. Its the case specially under neomycin selection.. Although we have not tested with other selection antiboitic but we never use neomycin after that finding. (http://www.ncbi.nlm.nih.gov/pubmed/23209606).. We also have shown there that due to the use of Neomycin global gene expression profile changes.... So be careful and plan your experiment accordingly.. I will suggest not to passage your cells for long as already suggested by Stefano..
Feel free to contact if you need to discuss any specific issue regarding this..
Good luck
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