用于构建RNA干扰载体的单寡核苷酸方法

One-oligonucleotide method for constructing vectors for RNA interference.

作者信息

Flores-Jasso Carlos Fabian, Velazquez-Quesada Ines, Landa-Solis Carlos, Gutierrez Andres A, Vaca Luis

机构信息

Departamento de Biologa Celular, Instituto de Fisiología Celular, Universidad Nacional Autóma De México, Ciudad Universitaria, Mexico City 04510, Mexico.

出版信息

Acta Pharmacol Sin. 2005 Dec;26(12):1467-73. doi: 10.1111/j.1745-7254.2005.00230.x.

DOI:10.1111/j.1745-7254.2005.00230.x

PMID:16297345

Abstract

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 H1. 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.

摘要

目的

开发一种简便、快速、自动化且廉价的方法，用于构建用于RNA干扰（RNAi）研究的短发夹RNA（shRNA）盒。

方法

使用单链寡核苷酸，以自动化方式在短短几分钟内构建了多种用于RNAi载体的DNA盒。将靶向增强绿色荧光蛋白（eGFP）的盒克隆到由RNA聚合酶III启动子H1驱动的质粒中。然后，将这些质粒转染到HeLa细胞中，随后用编码eGFP基因的重组腺病毒感染这些细胞。通过共聚焦成像和流式细胞术评估eGFP荧光水平。