What is the best possible method to knockout genes fro a primary DRG neuronal culture? ..Apart from extracting DRGs from a knockout mouse..Since a knockout mouse is unavailable for the gene that I am studying..
I see.. but if understand the principle correctly, how would CRISPR work in primary neuronal culture since neurons are non dividing?
Hi Divija,
For CRISPR to "work" cells do not need to divide. The problem with CRISPR in primary neuronal culture is the relatively low efficiency of transfection plus the uncertainty of having a partial or complete disruption of the gene (only one or both alleles affected). That's why knockdown the gen transcript by shRNA, RNAi or miRNA is the most common choice.
Cheers,
Thanks Sebastian,
What would you say is the % efficiency of shRNA knockdown vs CRISPR in primary neuronal cells ?
Hi Divija,
The actual knockdown efficiency will depend on how good is your RNAi/shRNA and the turnover of the protein (proteins with a low turnover will take long to be depleted even if the transcript is efficiently silenced). DRGs are quite difficult to transfect via liposome-based approach like lipofectamine (ca 5%). Nuclofection will give you, normally, a better efficiency (ca 25-50%).
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