Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
Front Immunol. 2022 Jul 6;13:918636. doi: 10.3389/fimmu.2022.918636. eCollection 2022.
Current genetic tools designed to target macrophages often target cells from all myeloid lineages. Therefore, we sought to generate a novel transgenic mouse which has a tamoxifen inducible Cre-recombinase under the control of the human CD68 promoter (hCD68-CreERT2). To test the efficiency and specificity of the of Cre-recombinase activity we crossed the hCD68-CreERT2 mice with a loxP-flanked STOP cassette red fluorescent protein variant (tdTomato) mouse. We established that orally dosing mice with 2 mg of tamoxifen for 5 consecutive days followed by a 5-day induction period resulted in robust expression of tdTomato in CD11b F4/80 tissue resident macrophages. Using this induction protocol, we demonstrated tdTomato expression within peritoneal, liver and spleen macrophages and blood Ly6C monocytes. Importantly there was limited or no inducible tdTomato expression within other myeloid cells (neutrophils, monocytes, dendritic cells and eosinophils), T cells (CD4 and CD8) and B cells (CD19). We also demonstrated that the level of tdTomato expression can be used as a marker to identify different populations of peritoneal and liver macrophages. We next assessed the longevity of tdTomato expression in peritoneal macrophages, liver and splenic macrophages and demonstrated high levels of tdTomato expression as long as 6 weeks after the last tamoxifen dose. Importantly, hCD68-CreERT2 expression is more restricted than that of LysM-Cre which has significant expression in major myeloid cell types (monocytes and neutrophils). To demonstrate the utility of this novel macrophage-specific Cre driver line we demonstrated tdTomato expression in recruited CD11bCD64F4/80 monocyte-derived macrophages within the atherosclerotic lesions of AAV8-mPCSK9 treated mice, with limited expression in recruited neutrophils. In developing this new hCD68-CreERT2 mouse we have a tool that allows us to target tissue resident macrophages, with the advantage of not targeting other myeloid cells namely neutrophils and inflammatory monocytes.
目前设计用于靶向巨噬细胞的遗传工具通常靶向所有髓系谱系的细胞。因此，我们试图生成一种新型转基因小鼠，该小鼠在人 CD68 启动子（hCD68-CreERT2）的控制下具有可诱导的 tamoxifen 诱导型 Cre 重组酶。为了测试 Cre 重组酶活性的效率和特异性，我们将 hCD68-CreERT2 小鼠与loxP 侧翼的 STOP 盒红色荧光蛋白变体（tdTomato）小鼠杂交。我们确定，连续 5 天每天给小鼠口服 2mg tamoxifen，然后进行 5 天诱导期，导致 CD11b F4/80 组织驻留巨噬细胞中 tdTomato 的强烈表达。使用这种诱导方案，我们证明了 tdTomato 在腹膜、肝脏和脾脏巨噬细胞和血液 Ly6C 单核细胞中的表达。重要的是，在其他髓样细胞（中性粒细胞、单核细胞、树突状细胞和嗜酸性粒细胞）、T 细胞（CD4 和 CD8）和 B 细胞（CD19）中，诱导性 tdTomato 表达有限或不存在。我们还证明，tdTomato 表达水平可用作识别腹膜和肝脏巨噬细胞不同群体的标志物。我们接下来评估了腹膜巨噬细胞、肝脏和脾脏巨噬细胞中 tdTomato 表达的持久性，并证明在最后一次给予 tamoxifen 剂量后长达 6 周，tdTomato 表达水平很高。重要的是，hCD68-CreERT2 的表达比 LysM-Cre 更受限制，LysM-Cre 在主要髓样细胞类型（单核细胞和中性粒细胞）中有显著表达。为了证明这种新型巨噬细胞特异性 Cre 驱动线的实用性，我们证明了在 AAV8-mPCSK9 处理的小鼠动脉粥样硬化病变中募集的 CD11bCD64 F4/80 单核细胞衍生的巨噬细胞中 tdTomato 的表达，在募集的中性粒细胞中表达有限。在开发这种新型 hCD68-CreERT2 小鼠时，我们有了一种工具，可以靶向组织驻留巨噬细胞，其优点是不靶向其他髓样细胞，即中性粒细胞和炎症性单核细胞。
