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学科主题: 药学
题名:
A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery
作者: Zhou, Demin1,2; Zhang, Jing2; Wang, Cuiying2; Bliesath, Joshua R.2; He, Qiuchen2; Yu, Dehua2; Li-He, Zhang1; Wong-Staal, Flossie2
关键词: RNAi ; inducible system ; lentiviral vector ; siRNA packaging
刊名: RNA-A PUBLICATION OF THE RNA SOCIETY
发表日期: 2009-04-01
DOI: 10.1261/rna.985209
卷: 15, 期:4, 页:732-740
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology
类目[WOS]: Biochemistry & Molecular Biology
研究领域[WOS]: Biochemistry & Molecular Biology
关键词[WOS]: DOUBLE-STRANDED-RNA ; MAMMALIAN-CELLS ; GENE-EXPRESSION ; SECONDARY STRUCTURE ; FUNCTIONAL SIRNAS ; TRANSCRIPTS ; MECHANISMS ; THERAPY ; ELEGANS ; LIBRARY
英文摘要:

The lentiviral vector is a useful tool for delivery of hairpin siRNA (shRNA) into mammalian cells. However, the efficiency of this system for carrying double-stranded siRNA (dsRNA) has not been explored. In this study we cloned the two forms of siRNA-coding sequence, a palindromic DNA with a spacer loop for shRNA and a double-stranded DNA with opposing Pol III promoters for dsRNA, into lentiviral DNA vectors, and compared their viral vector production yields. Our results indicate that sharply lower titer vector was obtained for dsRNA while much higher titer vector was produced for shRNA, posing a fundamental concern whether siRNA-carrying viral RNA itself is an inherent target of RNAi. Further experimental analyses using packaging cells that either allow or do not allow siRNA transcription indicate that the shRNA-carrying viral RNA is resistant to RNAi but the viral RNA carrier for dsRNA is not, offering a linker of RNAi bias-target secondary structure that causes shRNA vector to evade RNAi degradation. More importantly, the poor yield of dsRNA vector production was restored when a novel packaging cell line was used that blocks the antisense strand from dsRNA duplexes. This method has important implications for the RNAi field, especially for those who are using lentiviral dsRNA and dsRNA libraries for various biological discovery and therapeutic interventions.

语种: 英语
所属项目编号: 20852001 ; 985-2-126-121 ; 20080104
项目资助者: National Natural Science Foundation of China ; "985" Project Foundation ; The Key Laboratory Grant
WOS记录号: WOS:000264280100021
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/64196
Appears in Collections:北京大学药学院_期刊论文

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作者单位: 1.Peking Univ, Sch Pharmaceut Sci, State Key Lab Nat & Biomimet Drugs, Beijing 100191, Peoples R China
2.iTherX Inc, San Diego, CA 92121 USA

Recommended Citation:
Zhou, Demin,Zhang, Jing,Wang, Cuiying,et al. A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery[J]. RNA-A PUBLICATION OF THE RNA SOCIETY,2009,15(4):732-740.
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