利用CRISPR/Cas9建立PIN1基因敲除的成体神经干细胞系

Establishment of neural stem cells line with PIN1 gene knockout by CRISPR/Cas9

目的:利用CRISPR/Cas9基因编辑技术敲除成体神经干细胞(neural stem cell,NSC)PIN1基因,建立PIN1基因敲除的成体神经干细胞系。方法:取8周龄C57BL/6小鼠脑室下区的脑组织进行体外培养;设计靶向小鼠PIN1基因的单导向RNA(single guide RNA,sgRNA),以PX330质粒为骨架,构建PIN1的Cas9打靶载体,转染小鼠的成体神经干细胞,通过puro筛选和测序鉴定获得PIN敲除的单克隆细胞;利用免疫荧光染色和Western blot测定PIN1蛋白表达水平,免疫荧光染色鉴定神经干细胞的特征性标志物巢蛋白(Nestin)的表达,Beta Ⅲ Tubulin免疫荧光染色鉴定神经干细胞分化为神经元的能力。结果:成功构建PIN1基因的Cas9/sgRNA表达载体,转染后获得PIN1敲除的神经干细胞克隆9个。免疫荧光染色及Western blot显示PIN1蛋白无表达,免疫荧光染色显示PIN1敲除的神经干细胞Nestin阳性,神经干细胞分化培养时可部分分化为BetaⅢTubulin阳性的神经元细胞。结论:CRISPR/Cas9基因编辑技术可实现对小鼠成体神经干细胞PIN1基因的编辑。在PIN1基因敲除后PIN1蛋白无表达,初步表型分析显示,神经干细胞仍表达特征性标志物Nestin,并具有向神经元分化的能力。

Objective:CRISPR/Cas9 gene editing technology was employed to knockout the PIN1 gene of adult neural stem cells(NSCs)and finally established a PIN1 gene knockout adult neural stem cell line.Methods:The tissue from the subventricular zone of 8-week-old C57BL/6 mice was dissected and cultured in vitro;single guide RNA(sgRNA)targeting PIN1 gene in mice was designed,and the PX330 plasmid was used as the skeleton to construct PIN1 Cas9 target vector.PIN-knockout monoclonal cells were obtained after transfection and puro screening.The expression level of PIN1 protein was verified by immunofluorescence and Western blot.The expression of nestin was used to identify the neural stem cell characteristics.The differentiation ability to neuronal lineage was determined by beta Ⅲ tubulin immunofluorescence.Results:Cas9/sgRNA expression vector of PIN1 gene was successfully constructed,and nine PIN1 knockout neural stem cells were cloned after transfection.Immunofluorescence and Western blot showed no expression of PIN1 protein,immunofluorescence showed positive expression of nestin in PIN1 knockout neural stem cells,and neural stem cells could differentiate into beta Ⅲ tubulin positive neurons in differentiation culture.Conclusion:CRISPR/Cas9 gene editing technology can realize the editing of PIN1 gene in mouse adult neural stem cells.After knock-out of PIN1 gene,PIN1 protein is not expressed,but neural stem cells still partially express the specific marker nestin and have the ability to differentiate toward neurons.