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骨髓来源巨噬细胞中有效地敲除floxed基因的新方案
何丽,熊思东,肖晖
0
(苏州大学生物医学研究院,苏州 215123;中国科学院上海巴斯德研究所,上海 200031)
摘要:
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LysM-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mTOR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LysM-cre/mTORfl/fl BMDMs与mTORfl/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。
关键词:  髓源巨噬细胞  LysM-cre  floxed 基因  基因敲除
DOI:10.13610/j.cnki.1672-352x.20150825.008
基金项目:国家自然科学基金项目(31070779)资助。
An improved method for efficiently deleting floxed genes in bone marrow-derived macrophages
HE Li,XIONG Sidong,XIAO Hui
(Institute of Biology and Medical Sciences, Soochow University, Suzhou 215123;Institute Pasteur, Shanghai Chinese Academy of Sciences, Shanghai 200031)
Abstract:
Given the conventional method used to generate bone marrow-derived macrophages (BMDMs) in vitro often resulted in inefficient deletion of floxed genes by LysM-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 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.
Key words:  BMDMs  LysM-cre  floxed gene  gene knockout

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