子宫内电击转染技术在神经元树突棘发育及神经纤维归巢研究中的应用

Study of dendritic spines and axonal projection in mouse cerebral cortex using in utero electroporation

目的:建立鼠胚子宫内电击转染技术体系(In utero electroporation,IUE),探究该技术在研究神经元发育包括树突棘形成及神经纤维归巢(轴突投射)中应用的可行性。方法:构建带pCAG启动子表达绿色荧光蛋白(Green fluorescent protein,GFP)的质粒,并将该质粒利用子宫内电击转染技术转染到孕期15.5 d(E15.5)鼠胚大脑皮质的新生神经元中。待小鼠出生后(P0)及第28 d(P28)用4%多聚甲醛活体灌注固定,振荡切片机切片,免疫荧光组织化学染色,激光共聚焦显微镜观察拍照。结果:在出生后P28小鼠被转染一侧的大脑皮质内有大量被GFP标记的细胞,呈现典型的神经元形态特征,从胞体发出大量树突,树突表面有大量大小不等、形态不一的小棘,被标记神经元的轴突通过胼胝体投射到对侧海马和大脑皮质。结论:子宫内电击转染技术能成功转染大脑皮质神经元,由CAG启动的GFP表达载体能够在被转染的神经元中长时程高效表达GFP,并标记被转染神经元的所有细胞结构包括树突、轴突及树突棘,该技术可用于研究各种不同基因在小棘发育及神经纤维归巢中的作用。

Objective：To investigate the development of neurons in cerebral cortex, including dendritic spines and axonal pro- jections in vivo with in utero electroporation （IUE）, Methods： IUE was performed to transfect the green fluorescent protein （GFP） with the cytomegalovirus immediate early enhancer and chicken β-actin promoter fusion（CAG） promoter into the newborn neurons of embryonic mouse brains at embryonic day （E） 15.5. After perfused intracardially with ice-cold 4% paraformaldehyde （PFA） at post- natal day （P） 0 and P28, brains were sectioned on a vibratome. Sections were processed by immunocytochemical and fluorescence staining, the results were observed with eonfocal laser scanning microscope. Results： A large number of neurons were labeled with GFP proteins, and clearly seen at P28 after electroporation. The GFP positive cells presented a typical neuron morphological character- istics, abundance of dendrites with dendritic spines of different sizes and shapes on the surface grow out of the cell body of the neuron. The positive axons were slender and their collaterals extended in the hippocampus and the contralateral cerebral cortex acrossing the corpus callosum. Conclusion： The neurons can be transfected successfully with IUE. Long-term expression of the GFP cDNA plasmid with pCAG promoter visualized cardinal features defining the phenotype of cortical neurons, including axonal projection and the mor- phology of dendritic, axon and dendritic spine. This in utero electroporation method can be used for functional assays of genes in the development of dendritic spines and axonal projection.