一种新型荧光(mKate2)特异性标记中性粒细胞的转基因小鼠的构建及初步应用

Construction of a novel transgenic mouse model with mKate2 fluorescent protein-labeled neutrophils

目的构建在中性粒细胞中特异性表达mKate2红色荧光蛋白的转基因小鼠。方法本研究将人工合成的lysozyme M启动子及mKate2基因序列构建至pmini Tol2系统中,通过显微注射法把线性化的转基因载体注射到C57BL/6J小鼠的受精卵后并将其移植至假孕鼠体内,制备转基因小鼠。本研究使用PCR鉴定lyz-m Kate2转基因小鼠的成功构建和通过荧光显微镜和流式分析等方法鉴定该模型小鼠中荧光蛋白mKate2的表达及特异性。结果外源lyz-mKate2转基因盒在C57BL/6J小鼠体内成功表达。在外周血中检测到标记红色荧光信号的中性粒细胞,而且70%左右的mKate2阳性细胞是中性粒细胞。激光诱导血栓模型中血栓块可观察到mKate2阳性细胞。结论本研究成功构建了mKate2特异性标记中性粒细胞的新型转基因小鼠并验证了其在血栓模型中的应用价值。

This study was performed to establish a mouse model that specifically expresses mKate2 in neutrophils. Synthetic gene cassette, including mouse lysozyme promoter and mKate2 fluorescent protein coding sequence, was cloned into pminiTol2 transgenic vector, which was then linearized and microinjected into fertilized eggs of C57BL/6 J mice, to construct transgenic mice. Transgenic mice were genotyped by PCR; blood cell expression of mKate2 were examined with flow cytometry and fluorescent microscopy. PCR analysis of tail tip DNA confirmed that genomic integration of the transgenic cassette was successful. About 70% florescence positive blood cells were neutrophils. The transgenic mice were subjected to a photochemical injury at the carotid artery to induce thrombosis, and red florescent labeled neutrophils were identified in the thrombi formed after the injury. In conclusion, a novel transgenic mouse with neutrophils specifically labeled with mKate2 fluorescent marker has successfully constructed and its value has validated in the thrombosis model.