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Appl Biochem Biotechnol. 2017 Feb;181(2):699-709. doi: 10.1007/s12010-016-2242-1. Epub 2016 Oct 11.

Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP.

Xu J1,2, Zhu W1,2, Guo Y1,2, Jiang C1,2, Hussain N1,2, Meng L3,4, Lu S5,6.

Author information

1
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, 710061, People's Republic of China.
2
Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi'an, Shaanxi, People's Republic of China.
3
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, 710061, People's Republic of China. mengliesu@mail.xjtu.edu.cn.
4
Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi'an, Shaanxi, People's Republic of China. mengliesu@mail.xjtu.edu.cn.
5
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, 710061, People's Republic of China. lushemin@mail.xjtu.edu.cn.
6
Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Xi'an, Shaanxi, People's Republic of China. lushemin@mail.xjtu.edu.cn.

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.

KEYWORDS:

Lentivirus package; RNAi; TurboGFP; pLKO.1

PMID:
27730520
DOI:
10.1007/s12010-016-2242-1
[Indexed for MEDLINE]

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