miRNA慢病毒表达载体的构建及其在原代培养的大鼠胚胎大脑皮层神经细胞中的稳定表达

Construction and stable expression of miRNA lentivirus expression vector in primary cultured rat embryos cortical neurons

目的:构建miRNA的慢病毒表达载体,使其在原代培养的大鼠胚胎大脑皮层神经细胞中稳定表达。方法:将该干扰片段构建入慢病毒载体(pL KD-Ubc-eG FP-U6-shRNA)的U6启动子下游,该载体可以实现在干扰小片段RNA的同时表达绿色荧光蛋白EGFP基因,便于观察载体工作状态。通过脂质体转染法转染原代培养的大鼠胚胎大脑皮层神经细胞,观察转染的原代培养的大鼠胚胎大脑皮层神经细胞的形态及表达情况。结果:针对筛选的miRNA构建的慢病毒载体,经过DNA测序结果和琼脂糖凝胶电泳鉴定成功构建了筛选的miRNA的慢病毒表达载体,重组质粒转染原代培养的大鼠胚胎大脑皮层神经细胞,经24 h后在荧光倒置显微镜下可以观察到部分细胞带有绿色荧光,筛选的miRNA构建的慢病毒载体在原代培养的大鼠胚胎大脑皮层神经细胞中稳定表达。结论:筛选的miRNA的慢病毒表达载体构建成功,并稳定的转染到原代培养的大鼠胚胎大脑皮层神经细胞中,为miRNA对神经细胞存活功能的研究提供了平台。

Objective： To construct miRNA lentivirus expression vector, and keep stable expression in primary cul- tured cortical neurons. Methods： The interference fragment was cloned into downstream of the U6 promoter in the lenti- virus vector （pLKD-Ubc-eGFP-U6-shRNA）, which expressing cloned interference fragment and green fluorescent protein at the same time for the convenience of observing the working status of constructed miRNA lentivirus expression vector. The method of Liposome transfection was used to transfect primary cultured neurons, which were observed morphologicaly with inverted fluorescence microscope. Results： The miRNA-lentiviral vector construct was confirmed after DNA sequen- cing and agarose gel electrophoresis. After the recombinant plasmid transfected primary cultured neurons on 24 h, the transfeeted primary cultured neurons were observed to have green fluorescence under the inverted fluorescence micro- scope,and shown stable expression in primary cultured rat cortical neurons. Conclusion： The lentiviral vectors-miRNA was successfully constructed and stably expressed in the primary cultured neurons, which provided a new platform for the researchers to study miRNA function in neuron survival.