I need to replicate an experiment in which I used bone marrow-derived dendritic cells (BMDC) from cathepsin B-deficient mice (Ctsb-/-, in a C57BL/6 background) to target mice in a Balb/c background. The thing is, we don't have Ctsb-/- Balb/c, and due to our time/funding limitations, we can't generate them in a short period of time. Inhibitors are out of the question, because all the ones I've tried are never 100% specific for only Ctsb, and not for other cathepsins.
So, we are thinking about using siRNA targeting Ctsb (in both WT C57BL/6 and Balb/c) and see if we can replicate our results. I have a really nice protocol for using siRNA (for a different target) in BMDC; the thing is, freshly generated BMDC already have a basal level of expressed Ctsb (which is relatively abundant), so if I use siRNA with cells at this point, I would only knock down any de novo transcribed Ctsb, but can't do anything about the one that is already there.
I am thinking about using siRNA in bone marrow stem cells to target Ctsb, and then differentiate them into BMDC (with the Ctsb-/- mice, I already confirmed that Ctsb is dispensable for the differentiation of stem cells to functional BMDC).
Does anybody have any experience with something similar? Does anyone have tips on differences between using siRNA in bone-marrow stem cells vs. BMDC?
Hello,
In my experience You might advice use Lipofectamine RNAiMAX, which is specially designed for siRNA, and buy silencer select siRNA Ambion of the mark. The concentration of siRNA depends on what you want and silence, if just at the gene or protein level. If it is a gene level I suggest a concentration of 25 nM and a time of 24 hours, if 50 nM protein and 48h. Respect to concentration of Lipofectamine, this takes a manual recommends you, but is usually between 2-4 microliters per well, not much more because it is toxic.
I hope to be of help to,
Sincerely Santi.
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