Guo Yang, Zhu Mengyan, Yu Zhilan, Li Qing, Chen Yanjuan, Ci Lei, Sun Ruilin, Shen Ruling
Model Organism R&D Department, Shanghai Laboratory Animal Research Center, Shanghai, China.
Shanghai Engineering Research Center for Model Organizations, Shanghai Model Organisms Center, Inc., Shanghai, China.
Front Immunol. 2025 Mar 10;16:1482070. doi: 10.3389/fimmu.2025.1482070. eCollection 2025.
The new targeted gene editing technologies, such as the CRISPR/Cas system, enable researchers to insert or delete genes at targeted loci efficiently. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity.
Using the CRISPR/Cas9 system, we inserted the transgene expression cassette into the gene locus to generate conditional Cre-driver line knock-in mice, which drove the expression of CreERT2 by the endogenous promoter. By mating the strain with a reporter mouse strain which contains a tdTomato expression fragment blocked with a loxP-flanked STOP cassette (LSL) driven by a CAG promoter, a reporter strain was obtained to evaluate the expression pattern of CD2 in different cell types.
After treatment with tamoxifen, the knock-in mice were induced to perform efficient recombination at the site following CreERT2 activation and cause the expression of tdTomato fluorescence. The tdTomato and CD2 were expressed in the T cells of peripheral blood, spleen and mesenteric lymph nodes, whereas detected in a low proportion in the B cells. While about 20% of cells labeled with tamoxifen-induced tdTomato were CD2 monocytes in peripheral blood, 10% of dendritic cells were tdTomato/CD2 cells. Tamoxifen-independent expression of tdTomato occurred in approximately 3% of CD2 macrophages, but in negligible (~0.5%) in CD2 granulocytes.
This work supplied a new transgenic mouse as a valuable tool for lineage tracing in CD2-expressing cells, for conditional mutant studies of immune modulatory effects in a time-dependent manner, and analysis of the potential therapeutic effect of CD2-targeting biologics.
新的靶向基因编辑技术，如CRISPR/Cas系统，使研究人员能够在靶向位点高效地插入或删除基因。Cre-loxp重组系统被广泛用于以高时空特异性激活或失活基因。
利用CRISPR/Cas9系统，我们将转基因表达盒插入基因位点，以生成条件性Cre驱动系敲入小鼠，其通过内源性启动子驱动CreERT2的表达。通过将该品系与一个报告基因小鼠品系交配，该报告基因小鼠品系包含一个由CAG启动子驱动的、被loxP侧翼的STOP盒（LSL）阻断的tdTomato表达片段，获得了一个报告基因品系，以评估CD2在不同细胞类型中的表达模式。
用他莫昔芬处理后，敲入小鼠在CreERT2激活后在该位点被诱导进行高效重组，并导致tdTomato荧光表达。tdTomato和CD2在外周血、脾脏和肠系膜淋巴结的T细胞中表达，而在B细胞中检测到的比例较低。在外周血中，约20%用他莫昔芬诱导的tdTomato标记的细胞是CD2单核细胞，10%的树突状细胞是tdTomato/CD2细胞。tdTomato的非他莫昔芬依赖性表达约在3%的CD2巨噬细胞中出现，但在CD2粒细胞中可忽略不计（约0.5%）。
这项工作提供了一种新的转基因小鼠，作为在表达CD2的细胞中进行谱系追踪、以时间依赖性方式进行免疫调节作用的条件性突变研究以及分析靶向CD2的生物制剂潜在治疗效果的有价值工具。
