shRNA 资源库及其构建方法

1.NCBIshRNA资源库

About RNAi on CGAP

The NCBI is part of the consortium supporting the preparation of human and mouse libraries containing RNAi constructs that target cancer-relevant and other genes. The clones, prepared in the laboratory of Greg Hannon of Cold Spring Harbor, produce small RNA molecules, called shRNA (short hairpin RNA), that are available to the public from a commercial distributor.

The first shRNA clone release occured on March 25, 2004 and was in the SHAG-MAGIC vector. Recent modifications have generated an improved pSHAG-MAGIC2c vector and, therefore, the entire first set of clones has been replaced. This second release will contain targets to the original gene set and additional genes. However, the RNAi targets may be different from those in the first release due to the introduction of a new and improved algorithm. We hope to have the complete collection available by December 2005.

2.The RNAi Consortium shRNA Library

这个库已经分别被两家公司商品化，公开筛选序列，并请您可以购买它的整个库其中包括lentivirus library construct:reference to aritcle:Genome-scale loss-of-function screening　with a lentiviral RNAi library

2.1 Sigma-Aldrich

2.2 Open Biosystems

he RNAi Consortium, or TRC, is a public-private effort based at the Broad whose mission is to create a shRNA library as well to validate tools and methods that will enable the scientific community to use RNAi to determine the function of human and mouse genes. The reagents are composed of short hairpin sequences carried in lentiviral vectors arrayed in 96-well plates.

Library Distribution

# The TRC shRNA library is distributed as bacterial glycerol stocks, plasmid DNA or lentiviral particles by Sigma-Aldrich and as bacterial glycerol stocks by Open Biosystems. The Broad RNAi platform does not directly distribute the TRC library.

3.Hanna的RNAi库

hat is the RNAi Codex?

The impact of RNAi as a tool for mammalian biology is enormous and continues to grow. The goal of the RNAi Codex is to provide a single database that curates publicly available RNAi resources. Presently, the Codex describes clones from the Hannon-Elledge shRNA libraries (mouse and human) that are available through Open Biosystems. The Codex provides the most complete access to this growing resource, allowing investigators to see not only released clones but also those that are soon to be released. We also hope to incorporate information on other RNAi collections as they enter the public domain.Using the Codex, investigators can search for shRNA clones using several different approaches. For example, an accession number, a keyword, a locuslink ID, gene symbol or unigene cluster identifier can be provided. Alternatively, investigators can view clones that appear in functionally linked gene lists that have been hand-curated by experts in the relevant field. Finally a sequence or list of sequences can be provided and used to identify matching shRNAs in the collection.Links to descriptions of vector systems, protocols for clone use and commercial vendors from which the clones can be obtained are also provided.

方法：

3.1 PCR-based generation of shRNA libraries from cDNAs

Background: The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries;one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.Results: We report here an improved method for constructing genome-wide shRNA libraries enzymatically. The method includes steps of cDNA fragmentation and endonuclease MmeI digestion to generate 19-bp fragments, capping the 19-bp cDNA fragments with a hairpin oligonucleotide, and amplification of the hairpin structures by PCR. The PCR step converts hairpins into double-stranded DNAs that contain head-to-head cDNA fragments that can be cloned into a vector downstream of a Pol III promoter.Conclusion: This method can readily be used to generate shRNA libraries from a small amount of mRNA and thus can be used to create cell- or tissue-specific libraries.

3.2 Lessons from Nature: microRNA-based shRNA libraries

codex和Open Biosystems都应用的microRNA以及barcode高通量筛选模式

具体的内容参考：

http://www.screeninc.nl/gene/index.php

3.3 Methodology article

Criteria for effective design, construction, and gene knockdown by　shRNA vectorsThis article is available from: http://www.biomedcentral.com/1472-6750/6/7

3.4 An approach to genomewide screens of expressed small interfering RNAs in mammalian　cells PNAS 2004;101;135-140

3.5 A Simple and Economical Short-oligonucleotide-based Approach to shRNA GenerationJournal of Biochemistry and Molecular Biology, Vol. 39, No. 2, March 2006, pp. 329-334

3.6 Enzymatically prepared RNAi libraries

Large-scale RNA interference (RNAi) screens in mammalian cells have mainly used synthetic small interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries. The RNAi triggers for both of these approaches were designed with algorithm-based predictions to identify single sequences for mRNA knockdown. Alternatives to these approaches have recently been developed using enzymatic methods. Here we describe the concepts of enzymatically prepared shRNA and siRNA libraries, and discuss their strengths and limitations.