A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells.
Several vectors for the induction of RNA interference in mammalian cells have been described,based mainly on polIII-dependent promoters. They transcribe short hairpin RNAs (shRNA) that,after being processed into short interfering RNAs (siRNAs), mediate the degradation of the target mRNA. Here, we describe the construction of a new siRNA-expressing vector (psiUx) based on the strong and ubiquitous polII-dependent promoter of the human U1 small nuclear RNA (snRNA)gene. In psiUx, the only constraint for the shRNA sequence is a purine at position +1, since specific 3'-end formation is achieved by a box element located downstream of the transcribed region. Several constructs were designed against the lamin A/C target. Depending on the structure of the shRNA transcribed, a preferential or exclusive accumulation of the antisense strand is obtained, thus avoiding possible nonspecific targeting by the sense strand. In all cases tested, very effective siRNAs were produced, thus providing a proof-of-principle that a snRNA-type polII promoter can be used for the expression of siRNAs. We show that psiUx ensures high levels of expression and efficient knock down of the target gene also in stable cell lines.
Istituto Pasteur-Fondazione Centi-Bolognetti, Department of Genetics and Molecular Biology, University of Rome La Sapienza and IBPM of CNR, Rome, Italy.
Michela Alessandra
MA
Denti
Alessandro
A
Rosa
Olga
O
Sthandier
Fernanda Gabriella
FG
De Angelis
Irene
I
Bozzoni
D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A / Q000032:analysis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics*
D034904:Lamin Type A; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; C440308:lamin C; D012319:RNA Polymerase II