One-oligonucleotide method for constructing vectors for RNA interference.
OBJECTIVE
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%.


CONCLUSIONS
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.
Flores-Jasso
Carlos Fabian
CF
Departamento de Biologa Celular, Instituto de Fisiología Celular, Universidad Nacional Autóma De México, Ciudad Universitaria, Mexico City 04510, Mexico.
Velazquez-Quesada
Ines
I
Landa-Solis
Carlos
C
Gutierrez
Andres A
AA
Vaca
Luis
L
D001483:Base Sequence; D018390:Gene Targeting; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics*; D010957:Plasmids; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D009841:Oligonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins