Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33 % less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1+ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.

中文翻译：

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新型双顺反子 IL-3 报告小鼠忠实产生 IL-3 的评估。


白介素3（IL-3）是一种重要的造血生长因子和免疫调节细胞因子。尽管活化的 T 辅助细胞是 IL-3 的主要来源，但据报道其他细胞类型也表达这种细胞因子。然而，尚未对体内产生IL-3的细胞进行精确鉴定和定量。因此，我们使用 CRISPR/Cas 方法对含有双顺反子 mRNA 的小鼠进行工程改造，该双顺反子 mRNA 将易于识别的报告基因增强型绿色荧光蛋白 (ZsGreen1) 与 IL-3 表达连接起来。为了表征这些新型报告小鼠，我们首先检查了体外引发和激活的 CD4 T 细胞亚群的 ZsGreen1 表达。我们发现，与 Th0 和 Th2 细胞相比，活化的 Th1 细胞表达的 ZsGreen1 水平高出约 4 倍。尽管报告基因 Th1 细胞分泌的 IL-3 比类似激活的野生型细胞少约 33%，但内源性 IL-3 表达保持完整。为了表征报告小鼠在体内准确标记 IL-3 产生细胞的能力，我们用巴西圆线虫感染了小鼠。在初次和继发感染后，在肠系膜淋巴结和肺部检测到少量但显着数量的 ZsGreen1+ CD4 T 细胞。在受感染的报告小鼠和野生型小鼠之间没有观察到嗜碱性粒细胞和肠道肥大细胞数量的差异，这表明报告小鼠体内分泌IL-3水平，导致IL-3驱动的生物活性与在相应野生型小鼠中观察到的生物活性没有区别。型老鼠。这些 IL-3 报告小鼠将成为研究体内 IL-3 依赖性免疫反应的宝贵资源。