I am selecting for stable transformants in an N2a cell line. The construct looks like this: EF1a-gene of interest-IRES-eGFP...SV40-EM7-blasticidin resistance. I seem to be getting good selection, however the GFP signal seems to be diminishing over time and many of the cells seem to have lost it altogether despite growing well in blasticidin. Of course this is just by eye, but the signals are very reduced since first they were transfected. I am concerned about transcriptional suppression and would like to test this by using an inhibitor of some kind. Would HDAC inhibitors be useful? Should I use a different promoter perhaps? Other suggestions for selection?
Trichostatin A is a good one but expensive. Valproic acid is far less expansive. I have used both and they both have low toxicity. Try a few in a controlled experiment and discover if it makes a difference. You might also look at using histone methylase inhibitors.
Hi Michael,
You could try using a separate plasmid containing a different fluorophore and see if the fluorescence of the second plasmid also decreases. It could be a case of transcriptional repression but also could be a dilution of your plasmid with successive cell-divisions. Episomal plasmids tend to be lost over time.
You could try using a bi-cistronic plasmid to check for the transcriptional repression aspect. For the episomal problem, you could clone your gene into a piggybac plasmid and use transposase to insert it into the genome of N2a. It might be more stable that way.
I would direct you to a paper we published using the piggybac system and got excellent labelling. Garcia-Moreno et al., 2014
We would be happy to send you the piggybac plasmid alongwith the transposase if you need.
irreversible DNA methyltransferase inhibitors, such as 5-azacytidine and Decitabine, appears to be a promising option
Thanks guys, good suggestions. I'll give these a try and report back, but it may not be for a few weeks - grant season!
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