Hi,
I'm going to be transfecting some primary mammalian cells with a shRNA expressing plasmid in the next couple of days by electroporation. Reading up on the topic, I seem to find a number of investigators use Sodium butyrate to enhance expression of the transfected gene. One paper suggested that genes expressed under the SV40 promoter seemed to be particularly enhanced when added 24h after transfection, and could enhance insert expression up to five weeks later in stably transfected cells. The proposed mechanism as far as I can comprehend is that the butyrate acts as a HDAC inhibitor, maintaining the chromatin in an relatively unpacked state, facilitating expression. !!! YAY.....This is exactly what I needed!!!
While that was all very encouraging, I have also come across some papers where butyrate is used to achieve synchronization by cell cycle arrest (slightly higher conc's). This is a problem for me on two fronts: 1) I anticipate an anti-proliferative phenotypic consequence of knocking down the gene I'm targeting, and 2) being that my cells are primary, I only get a couple of population doublings before they become fibroblast-y and senescent. In this context, butyrate would probably slow proliferation as much if not more than my sh construct.
So....my questions are on the kinetics of this phenomena, as I'd like to get some ideas in terms of dosage and timing. Can the butyrate be used transiently? Any thoughts/experience on whether primary cells are refractory to butyrate treatment? What sort of dose range should I shoot for - and for how long - as the literature seems to be quite variable.
Ideally I would titrate this out and figure out an 'ideal' range, however the source tissue is exceedingly difficult to obtain, and quite laborious to extract the specific cells I need, so I'm hoping the forum might offer ideas. Cheers!-JAH
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