title: A new design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency.
abstract: BACKGROUND
  RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied.
  
  
  RESULTS
  To explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system.
  
  
  CONCLUSIONS
  We reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Jiening
authors.initials: J
authors.lastname: He
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Lian
authors.initials: L
authors.lastname: Huang
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Huiling
authors.initials: H
authors.lastname: Qiu
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Jiexuan
authors.initials: J
authors.lastname: Li
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Lan
authors.initials: L
authors.lastname: Luo
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Yanjiao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Shengli
authors.initials: S
authors.lastname: Tian
authors.affiliation: Department of Physiology, Shenzhen University Health Science Center, Shenzhen University, Shenzhen, 518060 Guangdong China.
authors.forename: Kang
authors.initials: K
authors.lastname: Kang
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, 712100 Shaanxi China.
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078 USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.affiliation: Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Nanhai Ave 3688, Shenzhen, 518060 Guangdong China.
authors.forename: Deming
authors.initials: D
authors.lastname: Gou
keywords: AGO2; Lentivirus; RNAi; shRNA