Seong Kyun Park1, Yun Kee2, Taehoon Ryu3, Hyoki Kim3, Byung Joon Hwang4. 1. Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University, Chuncheon, Kangwon-do, 24341, South Korea. 2. Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon, Kangwon-do, 24341, South Korea. 3. Celemics, 19F, Bldg.A, BYC Highcity, 131, Gasandigital 1-ro,Geumcheon-gu, Seoul, 153-718, Republic of Korea. 4. Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University, Chuncheon, Kangwon-do, 24341, South Korea. bjhwang@kangwon.ac.kr.
Abstract
BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. OBJECTIVE: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. METHODS: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. RESULTS: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. CONCLUSION: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.
BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. OBJECTIVE: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. METHODS: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. RESULTS: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. CONCLUSION: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.
Authors: Anton P McCaffrey; Leonard Meuse; Thu-Thao T Pham; Douglas S Conklin; Gregory J Hannon; Mark A Kay Journal: Nature Date: 2002-07-04 Impact factor: 49.962