Hi all,
I am trying to delete a protein of interest from postnatal cortical neuron cultures from floxed mice by introducting Cre via nucleofection (AMAXA system), but fail to see any loss of protein despite robust expression of the reporter (Cre-IRES-Cherry) in 60-70% of cells. I have used the same Cre plasmid to generate AAV that I can use to transduce the cultures and delete the protein, so it doesn't appear to be a promoter of DNA locus issue. I am using # of cells (8 million) and DNA amount (3 micrograms) that are the top recommended in the protocol, but perhaps I still need additional Cre DNA to get efficient recombination? Or is there something else that might be at play?
Hi Andrew, I would recommend 3 verifications: 1- Confirm the embryos used are homozygously floxed (I assume you do this routinely every time even when you have stable floxed parental lines). 2- Stain your cultures/blot them to confirm the expression of the recombinase. It may happens that the reporter is transduced to a higher extent than Cre and therefore you see red cells and very low recombination.
3- Ensure that the post-culture time and extend of Cre expression correspond with the turnover of the protein to analyze (high turnover will need less time to show decrease and vice versa).
Cheers,
Hi Sebastian,
Thanks for your answer. These 3 things are ones I have taken for granted based on how the experiment is set up - but definitely things to double check!
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