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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。
Objective To construct a transgenic mouse that specifically expressed mKate2 red fluorescent protein in neutrophils. Methods The synthetic lysozyme M promoter and mKate2 gene sequence were constructed into the pmini Tol2 system. The linearized transgene vector was injected into the fertilized egg of C57BL / 6J mice by microinjection and then transplanted into fake Pregnant mice, the preparation of transgenic mice. In this study, PCR was used to identify the successful construction of lyz-m Kate2 transgenic mice and to identify the expression and specificity of fluorescent protein mKate2 in this mouse model by fluorescence microscopy and flow cytometry. Results The exogenous lyz-mKate2 transgene cassette was successfully expressed in C57BL / 6J mice. Neutrophils labeled with a red fluorescent signal were detected in peripheral blood, and about 70% of mKate2-positive cells were neutrophils. MKate2 positive cells were observed in the thrombus mass in the laser induced thrombus model. Conclusion This study successfully constructed mKate2-specific neutrophils labeled transgenic mice and verified its value in thrombosis model.