Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP
Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP
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TurboGFP标记的便捷筛选pLKO.1-TRC载体的构建
DOI：
10.1007/s12010-016-2242-1
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发表时间：
2017-02-01
影响因子：
3
通讯作者：
Lu, Shemin
中科院分区：
文献类型：
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作者：
Xu, Jing;Zhu, Wenhua;Lu, Shemin
pLKO.1-TRC质粒由于其简单的操作和稳定性而成为流行和广泛使用的载体。基于该载体建立了一个庞大的RNAi数据库-TRC文库。然而，该质粒仅具有用于选择的嘌呤霉素抗性基因，这限制了其在显微镜和荧光激活细胞分选（FACS）中的应用。本研究在不改变原质粒结构的前提下，利用PCR、限制性内切酶酶切和分子克隆技术，将编码绿色荧光蛋白（GFP）的基因插入原质粒中，以扩大GFP的应用范围。为了证明新质粒的功能，我们在新构建的pLKO-TurboGFP-TRC（pLKOG）和原pLKO质粒的基础上构建了shNC和shGAPDH质粒，并通过293 T细胞包装慢病毒颗粒。收集含有慢病毒颗粒的上清液，然后与RAW 264.7细胞一起孵育用于感染。在使用嘌呤霉素选择7天后，收获细胞。RT-qPCR和Western blotting检测目的基因的表达。流式细胞仪检测细胞的绿色荧光。结果表明，构建的pLKOG质粒作为携带shRNA的慢病毒载体，能够有效地敲低目的基因的表达，并在宿主细胞中高效表达TurboGFP蛋白。结论：该质粒是一种方便的载体，可用于荧光显微镜和流式细胞仪筛选shRNA阳性细胞。
The pLKO.1-TRC plasmid has been a popular and widely used vector due to its simple handling and stability. The huge RNAi database, a TRC library, has been established based on this vector. However, this plasmid only has a puromycin-resisted gene for selecting, which limits its application in microscopy and fluorescence-activated cell sorting (FACS). In the present work, PCR, restriction endonuclease digestion and molecular cloning techniques were used to insert the gene decoding green fluorescent protein (GFP) without changing the structure of original plasmid to extend its application. To demonstrate the function of new plasmid, we constructed shNC and shGAPDH plasmids based on newly constructed pLKO-TurboGFP-TRC (pLKOG) and original pLKO plasmids, and then packaged lentivirus particles by 293T cells. The supernatant containing lentiviral particles was collected and then incubated with RAW264.7 cells for infection. After selection for 7 days by using puromycin, the cells were harvested. RT-qPCR and Western blotting were used to detect the target gene expression. FACS was used to detect the green fluorescent of cells. Our results showed that the newly constructed pLKOG plasmid, as a lentiviral vector carrying shRNA, could knock down the target gene expression efficiently and express TurboGFP protein efficiently in the host cells. We conclude that the new plasmid is a convenient vector for selecting positive cells with shRNA by using fluorescent microscope and FACS.