Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system.
RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking.
Kondo
Takefumi
T
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan.
Inagaki
Sachi
S
Yasuda
Kunio
K
Kageyama
Yuji
Y
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D002478:Cells, Cultured; D004251:DNA Transposable Elements / Q000235:genetics*; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000172:deficiency / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D005822:Genetic Vectors / Q000138:chemical synthesis*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D019076:Transgenes
D018528:ATP-Binding Cassette Transporters; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D005136:Eye Proteins; D034741:RNA, Small Interfering; C059254:w protein, Drosophila