title: Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules.
abstract: RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.
authors: Department of Microbiology, Immunology and Tropical Medicine, The George Washington University Medical Center, 2300 I Street, NW, Washington, DC 20037, USA.
authors: Mary A
authors: MA
authors: Ayuk
authors: Sutas
authors: S
authors: Suttiprapa
authors: Gabriel
authors: G
authors: Rinaldi
authors: Victoria H
authors: VH
authors: Mann
authors: Clarence M
authors: CM
authors: Lee
authors: Paul J
authors: PJ
authors: Brindley
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D023281:Genomics / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012550:Schistosoma mansoni / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D034741:RNA, Small Interfering; D008156:Luciferases