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Updated: Aug 14, 2026
DNA Vector-based RNA Interference to Study Gene Function in Cancer
Published on: June 4, 2012
One-oligonucleotide method for constructing vectors for RNA interference
Carlos Fabian Flores-Jasso1, Ines Velazquez-Quesada, Carlos Landa-Solis
1Departamento de Biologa Celular, Instituto de Fisiología Celular, Universidad Nacional Autóma De México, Ciudad Universitaria, Mexico City 04510, Mexico.
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.
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