Use of in vitro electroporation and slice culture for gene function analysis in the mouse embryonic spinal cord
Use of in vitro electroporation and slice culture for gene function analysis in the mouse embryonic spinal cord
复制标题
使用体外电穿孔和切片培养进行小鼠胚胎脊髓基因功能分析
DOI：
10.1016/j.mod.2019.103558
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发表时间：
2019-08-01
影响因子：
2.6
通讯作者：
Lin，Juntang
中科院分区：
文献类型：
--
作者：
Li，Shuanqing;Li，Yunxiao;Lin，Juntang
脊髓是中枢神经系统（CNS）的重要组成部分。目前，外源基因在胚胎小鼠脊髓中的表达需要子宫脊髓电穿孔，但这种技术的成功率很低。在这项研究中，我们通过结合胚胎小鼠脊髓体外电穿孔和器官脊髓切片培养两种方法，证明了外源基因在脊髓一侧的表达。取胚胎期12天的小鼠，将绿色荧光蛋白（pCAGGS-GFP）质粒注射到离体脊髓腔内，然后电穿孔。用振动切片机将脊髓切成300 μm的薄片。培养48 h后，脊髓一侧明显可见gfp阳性神经元，表明外源基因转染成功。轴突投射方向从脊髓内侧到脊髓外侧基本一致。与活体神经元相比，单个神经元在培养切片中具有更完整的神经突，有利于研究单个神经元的结构和行为变化。基于以上结果，我们成功建立了一种方便高效的外源基因在小鼠脊髓中的表达方法。
The spinal cord is an important part of the central nervous system (CNS). At present, the expression of the exogenous gene in the spinal cord of the embryonic mouse needsin uterospinal cord electroporation, but the success rate of this technique is very low. In this study, we have demonstrated the expression of an exogenous gene on one side of the spinal cord by combining two methods—in vitroelectroporation of embryonic mouse spinal cord and organ spinal cord slices culture. We took 12-day embryonic mice, injected the green fluorescent protein (pCAGGS-GFP) plasmid into the spinal cord cavityin vitro, and then electroporated. The spinal cord was cut into 300-μm slices using a vibratory microtome. After cultured for 48 h, GFP-positive neurons were clearly observed on one side of the spinal cord, indicating that the exogenous gene was successfully transferred. The axon projection direction is basically unanimous from the inside to the lateral edge of the spinal cord. Compared to neuronsin vivo, a single neuron in the culturing section has more complete neurites and is conducive to studying changes in the structure and behavior of individual neurons. Based on the above results, we have successfully established a convenient and efficient method for expressing the exogenous gene in the spinal cord of the mouse.