title: Discovery, adaptation and transcriptional activity of two tick promoters: Construction of a dual luciferase reporter system for optimization of RNA interference in rhipicephalus (boophilus) microplus cell lines.
abstract: Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vector of bovine babesiosis and anaplasmosis. Commercial promoters were evaluated for transcriptional activity driving luciferase expression in the tick cell lines. The human phosphoglycerate kinase (PGK) promoter resulted in detectable firefly luciferase activity within 2 days post-transfection of the R. microplus cell line BME26, with maximal activity at 5 days post-transfection. Several other promoters were weaker or inactive in the tick cells, prompting identification and assessment of transcriptional activity of the homologous ribosomal protein L4 (rpL4, GenBank accession no.: KM516205) and elongation factor 1α (EF-1α, GenBank accession no.: KM516204) promoters cloned from R. microplus. Evaluation of luciferase expression driven by various promoters in tick cell culture resulted in selection of the R. microplus rpL4 promoter and the human PGK promoter driving transcription of sequences encoding modified firefly and NanoLuc® luciferases for construction of a dual luciferase reporter system for use in tick cell culture.
authors.affiliation: USDA, Agricultural Research Service, Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, USA.
authors.forename: A P
authors.initials: AP
authors.lastname: Tuckow
authors.affiliation: USDA, Agricultural Research Service, Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, USA.
authors.forename: K B
authors.initials: KB
authors.lastname: Temeyer
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017930:Genes, Reporter; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D048494:Rhipicephalus / Q000166:cytology / Q000235:genetics*; D012269:Ribosomal Proteins / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D020648:Peptide Elongation Factor 1; D012269:Ribosomal Proteins; C056368:ribosomal protein L4; D008156:Luciferases
keywords: Ixodes; RNAi; Rhipicephalus; biochemistry; gene expression; luciferase; molecular genetics; parasitology; promoter; ticks; transcription