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A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery
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
2009-04-01
DOI10.1261/rna.985209
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
资助者National Natural Science Foundation of China ; "985" Project Foundation ; The Key Laboratory Grant ; National Natural Science Foundation of China ; "985" Project Foundation ; The Key Laboratory Grant
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
资助者National Natural Science Foundation of China ; "985" Project Foundation ; The Key Laboratory Grant ; National Natural Science Foundation of China ; "985" Project Foundation ; The Key Laboratory Grant
WOS记录号WOS:000264280100021
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被引频次:7[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/64196
专题北京大学药学院
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
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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.
APA Zhou, Demin.,Zhang, Jing.,Wang, Cuiying.,Bliesath, Joshua R..,He, Qiuchen.,...&Wong-Staal, Flossie.(2009).A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery.RNA-A PUBLICATION OF THE RNA SOCIETY,15(4),732-740.
MLA Zhou, Demin,et al."A method for detecting and preventing negative RNA interference in preparation of lentiviral vectors for siRNA delivery".RNA-A PUBLICATION OF THE RNA SOCIETY 15.4(2009):732-740.
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