Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Dec 8:5:67.
doi: 10.1186/s13578-015-0058-2. eCollection 2015.

A new design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency

Jiening He #  1 , Lian Huang #  1 , Huiling Qiu #  1 , Jiexuan Li  1 , Lan Luo  1 , Yanjiao Li  1 , Shengli Tian  1 , Kang Kang  2 , Jun Luo  3 , Lin Liu  4 , Deming Gou  1
Affiliations

A new design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency

Jiening He et al. Cell Biosci. .

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.

Keywords: AGO2; Lentivirus; RNAi; shRNA.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Effects of AGO1, AGO2, DICER or XPO5 overexpression on gene silencing. a Comparison of the silencing activities of two shRNAs against EGFP. HEK293 cells were co-transfected with pENTR/CMV-EGFP, pDsRed2-c1 and U6 promoter-driven shEGFP450, shEGFP417 or an unrelated shCon. Two days after transfection, the cells were then collected for flow cytometry analysis to evaluate EGFP knockdown efficiency by determining green to red (G/R) fluorescence intensity ratio; b, c effects of AGO1, AGO2, Dicer or XPO5 overexpression on the silencing of EGFP with shEGFP417 or shEGFP450. HEK293 cells were co-transfected with shRNA plasmid (shEGFP450, shEGFP417 or shCon), pENTR/CMV-EGFP, pDsRed2-c1 and plasmid expressing AGO1, AGO2, Dicer, XPO5 or empty control vector. Two days after transfection, G/R fluorescence intensity ratio was analyzed by flow cytometry and normalized to the shCon/control group; d representative images from the cells 2 days after transfection of pENTR/CMV-EGFP, pDsRed2-c1 and shGFP450 plasmids in the presence of AGO1, AGO2, Dicer, XPO5 or empty control vector; e G/R fluorescence intensity ratio measured from the cells transfected with shRNAmiR30a-based shEGFP417 (shEGFP417-miR30a) and the plasmid expressing AGO1, AGO2, Dicer, XPO5 or empty vector control. The results shown are mean ± standard deviation (SD) from three independent experiments. *P < 0. 05, ***P < 0.001
Fig. 2
Fig. 2
Dose-dependent analysis of AGO2 overexpression on RNAi efficiency. a Dose-dependent effects of AGO2 overexpression on EGFP silencing. HEK293 cells were co-transfected with pENTR/CMV-EGFP, pDsRed2-c1, shEGFP450 and different amount of AGO2 overexpression vector or the control vector without AGO2 insert. Representative images were shown from the cells 2 days after transfection; b the effect of AGO2 overexpression on EGFP silencing activities was quantified by analyzing G/R fluorescence intensity ratio. The results shown are mean ± SD from four independent experiments. ***P < 0.001 vs. control
Fig. 3
Fig. 3
Effect of stem length, loop size, and antisense strand location of shRNA on RNAi efficiency. a Design of a set of shEGFP variants with different stem lengths. The stem lengths were indicated in the name of each shRNA. All the constructs were designed to target EGFP, starting at the position of 417 of EGPF with the stem sequence extending to the 3′ direction. The hairpin loop was the same (5′-TTCAAGAGA-3′) for all of the vectors; b comparison of the activities of shRNAs with different stem lengths in HEK293 cells. G/R fluorescence intensity ratio was measured from the cells co-transfected with pENTR/CMV-EGFP, pDsRed2-c1, shRNA vector with or without AGO2 overexpression vector. shCon/control group was used to normalize the silencing efficiency; c design of 19-bp hairpin stem of shEGFP417 at the orientation of Sense-Loop-Antisense (S-L-AS) or Antisense-Loop-Sense (AS-L-S) structure with three kinds of loop sequences, L1 (4-nt, TTCG), L2, (6-nt, CTCGAG) or L3 (9-nt, TTCAAGAGA); d comparison of the activities of S-L-AS and AS-L-S shRNA with different loop lengths in the presence or absence of AGO2 co-expression. ShCon/control group was used for the normalization of EGFP silencing activities. The results shown are mean ± SD from four independent experiments. ***P < 0.001
Fig. 4
Fig. 4
Co-expression of AGO2 enhances shRNA-mediated knockdown of DsRed2 florescence reporter. a Design of a set of shRNAs targeting DsRed2 coding region at different positions; b a schematic illustration of a shRNA lentiviral vector with CMV promoter-driven AGO2 (shRNA/AGO2). The corresponding control (shRNA/control) was generated by removing AGO2 fragment; c U6-driven shRNA with the co-expression of CMV-driven AGO2 or its control in the single plasmid were co-transfected with EGFP/DsReds plasmids into HEK293. Two days after transfection, R/G fluorescence intensity ratio was measured by flow cytometry analysis and the silencing efficiency was normalized to the shCon/control group. The results shown are mean ± SD from four independent experiments. ***P < 0.001
Fig. 5
Fig. 5
Co-expression of AGO2 enhances shRNA-mediated knockdown of endogenous genes. a Design of a set of shRNAs targeting human ID1 or P65 mRNA at different regions. The number indicates the initial position of siRNA in the coding region of genes; b a lentivirus harboring U6-driven shID1 and CMV-driven AGO2 or its control was used to infect HeLa cells twice and selected with puromycin for 1 week. Representatives of Western blots using anti-ID1 or anti-ID2 antibodies were shown. β-actin was used as a loading control; c HeLa cell proliferation after silencing of ID1. The same number of stably selected HeLa cells expressing shRNA with or without AGO2 protein were seeded on 96-well plates at a density of 1 × 104 cells per well. After 24 h culture, cell proliferation was determined. The results shown are mean ± SD from four independent experiments. *P < 0.05 vs shCon, ***P < 0.001 vs shCon; d Western blot showing the knockdown of P65 in HeLa cells with a lentivirus-based shRNA with or without AGO2 co-expression. shCon was used as a negative control. β-actin was used as a loading control
Fig. 6
Fig. 6
Enhancement of shRNA efficiency achieved by DOX inducible AGO2 overexpression lentiviral vector. a A schematic illustration of pTAIPz-shRNA lentiviral vector co-expressing U6-driven shRNA and inducible AGO2 under control of the tetracycline-inducible TETO6 promoter; b comparison of two target genes knockdown in A549 cells infected with lentivirus expressing shRNA with or without AGO2 induction. β-actin served as the loading control
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391(6669):806–811. doi: 10.1038/35888. - DOI - PubMed
    1. Perkel JM. RNAi therapeutics: the teenage years. Biotechniques. 2012;52(6):355–357. doi: 10.2144/000113871. - DOI - PubMed
    1. Maczuga P, Lubelski J, van Logtenstein R, Borel F, Blits B, Fakkert E, Costessi A, Butler D, van Deventer S, Petry H, et al. Embedding siRNA sequences targeting apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy. Mol Ther J Am Soc Gene Ther. 2013;21(1):217–227. doi: 10.1038/mt.2012.160. - DOI - PMC - PubMed
    1. Berns K, Hijmans EM, Mullenders J, Brummelkamp TR, Velds A, Heimerikx M, Kerkhoven RM, Madiredjo M, Nijkamp W, Weigelt B, et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature. 2004;428(6981):431–437. doi: 10.1038/nature02371. - DOI - PubMed
    1. Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, Gasser B, Kinsey K, Oppel S, Scheiblauer S, et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 2007;448(7150):151–156. doi: 10.1038/nature05954. - DOI - PubMed
-