shRNA文库:RNA干扰研究者的福音 Nature 428, 427 - 431 (25 March 2004); doi:10.1038/nature02370
A resource for large-scale RNA-interference-based screens in mammals PATRICK J. PADDISON1,*, JOSE M. SILVA1,*, DOUGLAS S. CONKLIN1,*,†, MIKE SCHLABACH2,†, MAMIE LI2, SHOLA ARULEBA1, VIVEKANAND BALIJA1, ANDY O'SHAUGHNESSY1, LIDIA GNOJ1, KIM SCOBIE1, KENNETH CHANG1, THOMAS WESTBROOK2,†, MICHELE CLEARY3, RAVI SACHIDANANDAM1, W. RICHARD MCCOMBIE1, STEPHEN J. ELLEDGE2,† & GREGORY J. HANNON1 1 Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA 2 Department of Biochemistry, Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA 3 Rosetta Inpharmatics, 12040 115th Avenue NE, Kirkland, Washington 98034, USA * These authors contributed equally to this work † Present addresses: Department of Biomedical Sciences, Center for Functional Genomics, University at Albany, East Campus, B342A, One University Place, Rensselaer, New York 12144-2345, USA (D.S.C.); Department of Genetics, Harvard Partners Center for Genetics and Genomics, Harvard Medical School Room 158D, NRB, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA (M.S., T.W. and S.J.E.) Correspondence and requests for materials should be addressed to G.J.H. (hannon@cshl.org) or S.J.E. (selledge@genetics.med.harvard.edu).
Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.
2004年3月30日,Open Biosystems公司正式宣布Expression Arrest™ Human & Mouse Short Hairpin RNA (shRNA) Libraries <http://www.openbiosystems.com/expression_arrest_shrna_libraries.php>对外发售。该Expression Arrest™ shRNA Libraries构建者们设计、合成、测序并克隆了上千个小分子发夹RNA,研究者只需在网上数据库中选择特定的shRNA克隆就可在近期内得到用于RNAi用的shRNA。
世界著名分子生物学实验室-------美国冷泉港实验室(CSHL)的Dr. Hannon设计、测序并克隆获得了大量的shRNA克隆,shRNA可以在常规的DNA载体上表达。Expression Arrest™ shRNA克隆可以通过转染方法进入细胞,或者也可以通过病毒介导(腺病毒或逆转录病毒)进入细胞。体外稳定转染可以构建持续的细胞系,持久抑制目标基因表达。 全球的研究或商业性的实验室目前正在大范围的使用RNAi技术评价基因的功能,而Expression Arrest™ shRNA文库的产生促进了人和小鼠的基因功能研究,具有非常重要的科研和应用价值。
Expression Arrest™ shRNA文库由Open Biosystems公司运作上市,目前人和小鼠的文库总共包括6,500个基因,每个基因包括1-4个shRNA克隆。这些基因主要是一些非常有应用前景的基因,其蛋白质成分有可能在将来的疾病治疗领域发挥作用。该文库正在发展壮大,Open Biosystems寄希望将来的文库能包括目前所有证实的人和小鼠基因,且每个基因包括6-10个shRNA克隆。
Expression Arrest™ shRNA文库的出现是大规模RNAi筛选技术的突破,科研工作者可以通过非常简单的方式直接从该文库中找到针对某特定基因的shRNA,无需耗时耗力的siRNA设计、合成和验证过程。
Benefits of shRNA vs. siRNA |
|
siRNA |
shRNA |
使用代价 |
高 |
相对较低 |
持久性 |
最多2周 |
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