Abstract
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.
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Acknowledgments
The authors would like to thank Dr. Yan Jidong and Wang Yuteng for kindly giving the pLKO.1-TRC plasmid and plenti-TurboGFP-Puro plasmids. And we also would like to thank Dr. Trono’s Lab for kindly giving the packaging and envelope plasmids, Addgene #12260 and #12259. This work was supported by grants from the National Natural Science Foundation of China (No.81170017, 81401289, 81302527 and 81273211).
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Xu, J., Zhu, W., Guo, Y. et al. Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP. Appl Biochem Biotechnol 181, 699–709 (2017). https://doi.org/10.1007/s12010-016-2242-1
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DOI: https://doi.org/10.1007/s12010-016-2242-1