Upon cloning of plasmid DNA in Chemical competent E-coli cells (having dcm and dam), followed by its isolation using mini prep and using this plasmid for transfection in 3T3 cells ( murine fibroblast cells). Can this methylation affect the transcription of genes on the plasmid because they are blocked by methylation?
you might be interested in the following papers
http://www.ncbi.nlm.nih.gov/pubmed/12710908
http://www.ncbi.nlm.nih.gov/pubmed/11479556
http://www.ncbi.nlm.nih.gov/pubmed/11162475
http://www.ncbi.nlm.nih.gov/pubmed/10639520
http://www.ncbi.nlm.nih.gov/pubmed/10385302
http://www.ncbi.nlm.nih.gov/pubmed/16479578
all the best!
Didier
Just be aware that 3T3 cells are not great for transient transfection. If you use something like a GFP reporter, you will definitely see transfected cells, but the frequency will be much lower than in other cells types. If you are transfecting miniprep DNA, that may also lower your transfection efficiency. Of course there are a lot of other reasons that protein expression or detection can be poor for a given plasmid. If you are testing a new construct, definitely consider using 293T or other highly transfectable line before trying 3T3 cells. As for the issue with methylation, its a good thing to think about. However, eukaryotic DNA methylation, along with certain histone modifications, is a factor that influences the formation of heterochromatin and gene silencing. This isn't an issue for transient transfection, and if you are doing stable transfection, DAM/DCM modifications won't occur. Certainly, RNA polymerase might be affected by plasmid methylation, but I just have never heard of anyone worrying about it. Thus, if you are getting poor expression in 3T3 cells, I would consider other issues first before trying to eliminate the methylation in E. coli.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry