One-oligonucleotide method for constructing vectors for RNA interference

• 来源期刊: 中国药理学报(英文版)
• 关键词: RNA interference shRNAs DNA cassette RNAi vector oligonucleotide
• 年，卷（期）: 2005,（12）
• 页码范围: 1467-1473
• 页数: 7页
• 语种: 英文

摘要

Aim: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. Methods: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP,were cloned into plasmids driven by RNA polymerase Ⅲ promoter H 1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. Results: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. Conclusion: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.