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KMID : 0381120190410050573
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Genes and Genomics
2019 Volume.41 No. 5 p.573 ~ p.581
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Enzymatic construction of shRNA library from oligonucleotide library
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Park Seong-Kyun
Kee Yun
Ryu Tae-Hoon
Kim Hyo-Ki
Hwang Byung-Joon
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Abstract
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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.
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KEYWORD
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RNAi, ShRNA library, Oligonucleotide library, High-throughput screen
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