I am looking for methods by which I can knock down the expression a protein in acute hippocampal slices using siRNA. First of all, is it possible to do so using siRNA in acute hippocampal slices? If yes then please tell me how to go ahead for that. If not, what are the alternative methods to do so? I have a chemical inhibitor for this purpose but I want to use siRNA based knockdown as a complementary experiment. Looking forward for your suggestions.
Hi,
I don't have a clear answer to this question, but there are many papers reported that knocked-down of protein expression in organotypic cultured slice system using RNA interference. How about using short-hairpin RNA (shRNA) which co-expresses GFP in an organotypic slice culture? You can also use non-targeting siRNA as a control (shNT).
To confirm the knockdown effect of (gene or protein of interest)-targeting shRNA, you can use lentiviral infection system with primary cultured hippocampal neurons.
I would only offer up my personal view from many years of work on cultured hippocampal slices. I recommend you to use the biolistic transfection methods (gene gun, i.e.). Hope this helps.
Yeah ... thanks a lot for your help.. thanks a lot... I too read papers on RNA interference and organotypics slice cultures.
So it does not seem to be feasible to apply siRNA or RNAi in "acute" slices? And just to clarify, the acute slices refer to the slices acquired from adult animals (rats, mice, etc) and can be maintained in room temperature for around 10 hours...
Any input would be appreciated!
Hi Tao
RNAi has been tried for some acute prep slice that we usually use. But it's tough, but new nanoparticle delivery is possible.
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