title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells.
abstract: Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral [Murine leukemia virus (MLV)] and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs-stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.
authors.affiliation: Department of Microbiology, Immunology & Tropical Medicine, The George Washington University Medical Center, Ross Hall 448, 2300 I Street NW, Washington, DC 20037, USA.
authors.forename: Raphaël
authors.initials: R
authors.lastname: Duvoisin
authors.forename: Mary A
authors.initials: MA
authors.lastname: Ayuk
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rinaldi
authors.forename: Sutas
authors.initials: S
authors.lastname: Suttiprapa
authors.forename: Victoria H
authors.initials: VH
authors.lastname: Mann
authors.forename: Clarence M
authors.initials: CM
authors.lastname: Lee
authors.forename: Nicola
authors.initials: N
authors.lastname: Harris
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brindley
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005354:Fibrosarcoma / Q000235:genetics / Q000378:metabolism*; D017238:Genes, Helminth; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000378:metabolism; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics*; D012550:Schistosoma mansoni / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D049409:Luciferases, Firefly