An improved method for efficiently deleting floxed genes in bone marrow-derived macrophages
Given the conventional method used to generate bone marrow-derived macrophages(BMDMs) in vitro often resulted in inefficient deletion of floxed genes by Lys M-cre, we attempted to improve this method to gain larger number of BMDMs with better deletion efficiency of floxed genes. By adjusting the schedule for medium change to maintain stable M-CSF concentration and using Trypsin to digest BMDMs, we were able to obtain two times more BMDMs(5×107 cells/per mouse) than the conventional protocol. Furthermore, our new protocol also resulted in improved deletion efficiency of floxed m TOR gene in BMDMs(76.9%±7.83% vs 36.77%±5.07%). Accordingly, we were able to detect an elevated IL-6 and IL-12 expression in Lys M-cre/m TORfl/fl BMDMs generated by the new protocol. Therefore, we reported here a new method to improve the quantity and gene deletion efficiency in BMDMs, which will be highly beneficial for studies using Lys M-cre to generate knockout cells and also improve animal welfare.