Hello all.
B cells isolated from chick embryos' bursa do not survive in culture for very long. (down to 30% viability after 9 hours of incubation). I really wanted to test the effect of demethylating the DNA on several aspects of that cell. Problem is, 5-azac works on "trapping" the enzymes in charge of methylating the DNA (DNMT), so the cells have to go through at least several cycles for that material to take a proper effect. Protocols I have seen use 5-azac on cultures of cell lines for a minimum of 24 hours, and most of them use it for 3 days. This is not an option for bursal B cells. Does anyone have any experience with faster acting demethylators?
Thank you
This is a difficult problem, because you are dealing with two separate issues, first, you need to get drug incorporation in S phase cells, second, you need to re-pattern DNA methylation, which is also largely replication dependent. My only suggestion would be to try 5-aza-2-deoxycytidine (decitabine), not 5-azacytidine, because you will avoid the need for ribonucleotide reductase conversion of the drug, and should get faster DNA incorporation with decitabine. Depending on whether the cells replicate or not, you could observe some small degree of DNA demethylation. Other putative agents that may act more directly on DNMT enzymes would still have similar issues for re-patterning DNA methylation. Decitabine is very potent and is probably worth a try.
Both 5-aza and Decitabine are drugs that work in vivo. You can treat your hens with them before isolating the embryos, if it's worth the effort. Dosage, treatment schedules, and delivery routes can be determined from clinical trials in humans and previous studies, e.g. http://www.ncbi.nlm.nih.gov/pubmed/6204332
Also, some in vitro work that may be relevant: http://www.ncbi.nlm.nih.gov/pubmed/22438967
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