神经系统SARM1条件性敲除小鼠的构建及其应用

目的：构建SARM1基因在神经细胞中条件性敲除小鼠模型，为探究SARM1基因在神经系统中的功能提供研究工具。方法：运用ES细胞打靶的方式构建SARM1flox/flox转基因小鼠，并将其与表达Nestin-Cre重组酶的工具鼠进行杂交以产生神经元特异性SARM1基因敲除小鼠。然后使用PCR对敲除小鼠进行基因型鉴定，使用Western blot验证基因敲除效果。通过对小鼠进行旷场与高架十字迷宫试验来评估小鼠的情绪行为和焦虑水平。结果：SARM1蛋白主要表达于中枢神经系统，在神经元中高表达。PCR结果表明，成功构建了神经元特异性SARM1基因敲除小鼠。Western blot结果显示，与正常小鼠对比，SARM1Nestin-CKO小鼠的脑组织中SARM1蛋白表达显著降低（P＜0.05），并且SARM1基因敲除不影响小鼠的正常生长发育，也不影响脑组织的一般形态和神经细胞的数量。同时发现该条件性敲除小鼠不存在明显的焦虑样表型。结论：成功构建了SARM1基因在神经细胞中特异性敲除的小鼠动物模型，可为探讨SARM1基因在神经精神疾病中的作用提供重要研究平台。

Construction of SARM1 conditional knockout mice in nervous system and its application

Objective: To construct neuronal-specific conditional SARM1 knockout mice and to provide a research tool for exploring the function of SARM1 in the nervous system. Methods: SARM1flox/flox transgenic mice were generated by using embryonic stem (ES) cell targeting technology. Next SARM1flox/flox transgenic mice were crossed with Nestin-Cre transgenic mouse to generate neuronal-specific conditional SARM1 knockout mice. After that the conditional SARM1 KO mice were genotyped by using polymerase chain reaction (PCR). Western blot was performed to confirm the loss of SARM1 protein in the brain tissues of these mice. Behavior tests such as open field and elevated plus maze test were performed to assess any emotional behavior and general anxiety level in these mice. Results: SARM1 protein was highly expressed in the central nervous system, which was mainly expressed in neurons. Genotyping results revealed that neuronal-specific conditional SARM1 knockout mice were successfully generated. Further more, compared with wild type mice, SARM1 protein level was significantly decreased in brain tissues of SARM1Nestin-CKO mice (P<0.05). Conditional knockout of SARM1 gene in central nervous system did not affect the growth and development of mice, or the general morphology of brain tissue and the number of neuronal cells. Meanwhile, these mice had no obvious anxiety disorder phenotype. Conclusion: We have successfully generated a mouse line that specifically knocks out the SARM1 gene in neuronal cells. The application of this mutant mouse line may provide a useful tool for exploring the role of SARM1 gene in neuropsychiatric diseases and neurodegenerative diseases.