骨髓来源巨噬细胞中有效地敲除floxed基因的新方案

An improved method for efficiently deleting floxed genes in bone marrow-derived macrophages

探索了在骨髓来源巨噬细胞(BMDMs)中有效地敲除floxed基因,更好地研究基因敲除引起的表型的新方案。由于传统方法往往导致floxed基因不能被Lys M-cre酶高效地敲除,阻碍了BMDMs在一些遗传学方面的应用。通过调整培养基的更换时间,及时补充新鲜的M-CSF,使用胰酶消化分盘,有效地提高了细胞得率,并增强了基因在BMDM中的敲除效率。与传统方法相比,新方案将BMDMs得率增加了1倍,每只小鼠能获得接近5×107个BMDMs。新方案也显著性地提高了巨噬细胞中m TOR基因的敲除效率,从36.77%±5.07%提高到了76.9%±7.83%。新方案获得的Lys M-cre/m TORfl/fl BMDMs与m TORfl/fl BMDMs相比,在应答LPS刺激方面出现了显著的表型差异,表达了更多的细胞因子IL-6和IL-12。在小鼠来源受限和需要提升巨噬细胞中特定基因敲除效率的情况下,本方案具有极为突出的优势,用少量的小鼠获得可靠的实验数据,也符合动物实验的"3R"原则。

Given the conventional method used to generate bone marrow-derived macrophages （BMDMs） in vitro often resulted in inefficient deletion offloxed genes by LysM-cre, we attempted to improve this method to gain larger number of BMDMs with better deletion efficiency offloxed 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 ×10^7 cells/per mouse） than the conventional protocol. Furthermore, our new protocol also resulted in improved deletion efficiency of floxed mTOR 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 LysM-cre/mTORfl/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 LysM-cre to generate knockout cells and also improve animal welfare.