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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 발행연도
- 2019.1
- 수록면
- 573 - 581 (9page)
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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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참고문헌 (27)
참고문헌 신청
Baum JA / 2007 / Control of coleopteran insect pests through RNA interference / Nat Biotechnol 25:1322~1326
Bernstein E / 2001 / Role for a bidentate ribonuclease in the initiation step of RNA interference / Nature 409:363~366
Castanotto D / 2005 / Targeting cellular genes with PCR cassettes expressing short interfering RNAs / Methods Enzymol 391:173~185
Catalanotto C / 2000 / Gene silencing in worms and fungi / Nature 404:245
Donzé O / 2002 / RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase / Nucleic Acids Res 30:46
Bernstein E / 2001 / Role for a bidentate ribonuclease in the initiation step of RNA interference / Nature 409:363~366
Castanotto D / 2005 / Targeting cellular genes with PCR cassettes expressing short interfering RNAs / Methods Enzymol 391:173~185
Catalanotto C / 2000 / Gene silencing in worms and fungi / Nature 404:245
Donzé O / 2002 / RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase / Nucleic Acids Res 30:46
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