腺相关病毒介导的小鼠发育期大脑中的特定细胞和特定脑区的基因编辑及潜在应用探索

Adeno-associated virus mediated cellular and regional specific gene editing and the potential applications in the developmental brain of mice

目的探索腺相关病毒(AAV)用于发育期星形胶质细胞的高分辨稀疏标记以及在皮质和海马中的区域性标记的可能性。方法在成年小鼠海马立体定位注射携带有绿色荧光蛋白(EGFP)的AAV5、AAV8和AAV9,比较感染效率。然后,立体定位注射AAV5和AAV9至新生小鼠大脑皮质和海马,观察稀疏细胞标记和区域性标记的标记效果。结果三种血清型AAV的感染效率依次为AAV8> AAV9> AAV5,AAV5及AAV8分别对星形胶质细胞和神经元具有较高亲嗜性。AAV5注射至P0小鼠皮质,在P9时即可实现稀疏标记,且标记信号强度高,高分辨激光共聚焦显微镜下3D扫描后适用于星形胶质细胞的形态3D重建和量化分析。通过对P0小鼠的立体定位注射,还实现了发育期小鼠皮质和海马的区域性基因转导。结论根据感染效率和细胞亲嗜性选择AAV血清型,通过立体定位注射,能够实现发育期小鼠特定细胞类型和脑区的高效基因转导。上述结果有助于扩展AAV在中枢系统发育和相关疾病中的应用范畴。

Objective To explore the possibility of astrocytic high resolution sparse labeling and regional labeling of the hippocampus and cortex in the developmental brain of mice,by using AAVs.Methods AAV5,AAV8 and AAV9 packaging with enhanced green fluorescent protein(EGFP)were stereotactic injected into the hippocampus of adult mice,and the transfection efficiency of each AAV serotype was compared.Thereafter,AAV5 and AAV9 were stereotactic injected into the cortex and hippocampus of postnatal(P0)mice,accessing the efficiency of astrocytic high resolution sparse labeling and regional labeling of hippocampus and cortex.Results The transfection efficiency of the three AAV serotype vary,i.e AAV8 >AAV9 >AAV5.AAV5 and AAV8 showed a high astrocytic and neuronal tropism,respectively.Stereotactic injection of AAV5 into the cortex of P0 mice resulted in a high resolution sparse labeling of astrocyte,as earlier at P9.After 3D scanning via laser confocal microcopy,the high resolution figures were suitable for astrocytic 3D reconstruction and morphological quantitative analysis.Furthermore,stereotactic injection of AAVs at P0 also achieved effectively regional genes delivery into the cortex and hippocampus of the developmental mice.Conclusion Stereotactic injection of the properly selected AAV according to their transfection efficiency and cellular tropism,can achieve cellular and regional specific genes delivery in the developmental brain of mice,powerfully.Our findings further expand the application of AAVs in the investigating of the central nerve system(CNS)development and the related diseases.