Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/6/2015
Publication Date: 8/1/2015
Publication URL: http://handle.nal.usda.gov/10113/62213
Citation: Tuckow, A.P., Temeyer, K.B. 2015. 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. Insect Molecular Biology. 24(4):454-456.
Interpretive Summary: The southern cattle tick transmits at least two fatal diseases to cattle and was eradicated from the United States but presents a continuing threat of re-entry and re-establishment in the U.S. Tick resistance to available acaricides has become widespread and research to develop new tick control methods is essential. Molecular biological approaches investigating novel targets for tick control through vaccines or new pesticides is essential to development of new tick control technology. New research reports the discovery of two functional promoter sequences from the southern cattle tick. Promoter sequences regulate gene expression, and the two new tick promoters are the first functional promoter sequences that have been identified from ticks. Promoters are valuable tools in molecular biology, and one of the new promoter sequences was used to construct a new and highly sensitive reporter plasmid that can be used to measure the efficiency of gene silencing for specific tick target genes in tick cell cultures. Gene silencing is a useful technique to "turn off" expression of a target gene in order to find out information about what the target gene does inside cells or organisms. The new method enables researchers to perform tick gene silencing experiments much faster and at greatly reduced cost. In addition to use in the new reporter plasmid, the promoter sequences will also enable expression of genes in tick cell culture to produce desired proteins allowing new discoveries about the functions and interactions of proteins within the cell or tick, thereby providing new information that will help identify new key targets for tick control.
Technical 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 PGK (phosphoglycerate kinase) 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 RPL4 and EF-1a 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.