A multi-shRNA vector enhances the silencing efficiency of exogenous and endogenous genes in human cells

Weng,Yanjie; Shi,Ying; Xia,Xi; Zhou,Wenjuan; Wang,Hongyan; Wang,Changyu

doi:10.3892/ol.2017.5672

A multi-shRNA vector enhances the silencing efficiency of exogenous and endogenous genes in human cells

Authors:
- Yanjie Weng
- Ying Shi
- Xi Xia
- Wenjuan Zhou
- Hongyan Wang
- Changyu Wang
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Affiliations: Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China
Published online on: February 2, 2017 https://doi.org/10.3892/ol.2017.5672
Pages: 1553-1562
Copyright: © Weng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

RNA interference (RNAi) is a powerful technology for suppressing gene function. In most studies, small interfering RNAs (siRNAs) consist of one short hairpin RNA (shRNA) and, therefore, are often unable to achieve loss‑of‑function of their target genes. In the current study, an RNAi vector containing three shRNAs under the control of three RNA polymerase III U6 promoters was constructed. RNAi vectors containing one or two shRNAs were generated for comparisons. A pilot study targeting exogenously expressed DsRed in the HEK293 cell line revealed promising effects and a high selectivity for the multi-shRNA RNAi vector. Akt2 is constitutively expressed in cultured SKOV3 human ovarian cancer cells, and the multi-shRNA RNAi vector showed a strong efficiency for downregulating the expression of Akt2 in these cells, with no apparent interferon response. In addition, the Akt2‑3shRNA vector, containing three shRNAs targeting Akt2, showed the best effect of all the shRNA vectors in reversing paclitaxel-induced resistance in SKOV3 cells. This study developed a widely applicable resource for enhancing the efficiency of gene silencing and a novel technique for performing complex loss-of-function screens in mammalian cells.

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