Hi!
I work with a mouse model that expresses the Cre recombinase, so that only upon tamoxifen stimulation the animals express Cre, thus I can induce the KO of the floxed gene. I know that in vitro people are using 4-hydroxytamoxifen in vitro (and it has already been used in microglia as well), but my problem is... I need the cells in suspension after tamoxifen. The reports I've seen using tamoxifen, state that they plated the cells and then did the tamoxifen stimualtion. My wuestion is:
Has anyone ever tried to induce the Cre recombinase with 4-hydroxytamoxifen when microglia are still on the flasks (before shaking them off)?
Thanks a lot!
Hi Amanda. We have always induced recombination whilst the microglia are still in the flask that is, as you have asked, before shaking them off. The catch is, it takes at least 5 to 7 days for the recomination to kick in and microglia may not be really happy for that long once they are taken off the astrocyte layer. That was my experience, at least. Good luck
Hi Phani!! Thank you so much! Which concentration of tamoxifen did you use?
Our concentration is 20mg/mL and we add it at 1:1000 to the cultures
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