Tuckow and Temeyer (2015) describe their isolation and evaluation of two promoters from the tick Rhipicephalus (Boophlus) microplus and their use of those promoters in a dual luciferase reporter system that enabled them to test the efficacy of dsRNAi constructs in the R. microplus cell line BME26 prior to their use in vivo.
R. microplus is an important vector of pathogens and is a veterinary pest throughout the world while in the United States, where it has been largely erradicated, one is only able to work on them in certain quarantined facilities. This can be challenging.
Tuckow and Temeyer review how RNAi-based gene silencing is the primary functional genomics tool available for this and other species of ticks. Because of the challenges of working with these insects the authors reasoned that having an in vitro system for testing RNAi-based silencing constructs would be beneficial. RNAi strategies could be validated in vitro before moving to more challenging in vivo studies.
To create such an in vitro system Tuckow and Temeyer create a dual luciferase expression system on a single plasmid into which one can clone your silencing target. Co-tranfecting dsRNAi expressing plasmids with the dual luciferase plasmid containing the target gene enabled them to validate their RNAi targets and dsRNAs they used to silence gene expression.
Before being able to create a functional dual luciferease system Tuckow and Temeyer assess a number of promoters for their activity in R. microplus cells and find that the promoter from human phosphoglycerate kinase was active while the promoter from SV40 was not active in the tick cell line BME26.
Tuckow and Temeyer isolate upstream flanking sequences associated with the genes elongation factor-1 alpha (EF-1α) and rpl4 and test their abilities to drive reporter gene expression in BME26. Both genes are expected to be constitutively expressed and while the authors do not report an extensive analysis of these promoters they conclude that rpL4 and EF-1α can be used to drive expression of two different secreted luciferase reporter gene constructs.
The rpl4 promoter fragment was 674 bp in length while that of EF-1α was 893 bp making these reasonably convenient to use.
With the incorporation of these promoters into a dual luciferase plasmid the authors feel the the dual reporter system they assembled was good for evaluating dsRNA, siRNA and miRNA in tick cells.
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 Mol Biol: doi: 10.1111/imb.12172.