Rapid construction of small interfering RNA-expressing adenoviral vectors on the basis of direct cloning of short hairpin RNA-coding DNAs.
In the conventional method for constructing an adenoviral (Ad) vector expressing small interfering RNA (siRNA), short hairpin RNA (shRNA)-coding oligonucleotides are introduced downstream of a polymerase III (or polymerase II)-based promoter cloned into a shuttle plasmid. An siRNA expression cassette, which is cloned into the shuttle plasmid, is then introduced into the E1 deletion region of the Ad vector plasmid by in vitro ligation or homologous recombination in Escherichia coli, and the linearized plasmid is transfected into 293 cells, generating an Ad vector expressing siRNA. Therefore, two-step plasmid manipulation is required. In this study, we developed a method by which shRNA-coding oligonucleotides can be introduced directly into the Ad vector plasmid. To do this, we constructed a new vector plasmid into which the human U6 promoter sequence was cloned in advance. Unique restriction enzyme sites were introduced at the transcription start site of the U6 promoter sequence in the vector plasmid. Luciferase and p53 genes were efficiently knocked down by Ad vectors generated by the new method and expressing siRNA against the target gene. This method should be useful for RNA interference-based experiments, and should make it easy to construct an siRNA-expressing Ad vector library for functional screening.
Mizuguchi
Hiroyuki
H
National Institute of Biomedical Innovation, Osaka 567-0085, Japan. mizuguchi@nibio.go.jp
Funakoshi
Naoko
N
Hosono
Tetsuji
T
Sakurai
Fuminori
F
Kawabata
Kenji
K
Yamaguchi
Teruhide
T
Hayakawa
Takao
T
D000256:Adenoviridae; D017498:Adenovirus E1 Proteins / Q000235:genetics; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics
D017498:Adenovirus E1 Proteins; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D016159:Tumor Suppressor Protein p53; C405486:U6 small nuclear RNA