,One-oligonucleotide method for constructing vectors for RNA interference

• 作 者: Carlos Fabian FLORES-JASSO Ines VELAZQUEZ-QUESADA Carlos LANDA-SOLIS Andres A GUTIERREZ Luis VACA
• 机 构: Departamento de Biología Celular, Instituto de Fisiología Celular, Universidad Nacional Autónoma De
• 出 处: 中国药理学报(英文版)
• 发表日期: 2005年12期
• 关键词: RNA interference shRNAs DNA cassette RNAi vector oligonucleotide

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.