Development and validation of a dual luciferase reporter system for in vitro evaluation of gene silencing efficacy in the Southern cattle tick: Comparison to in vivo gene silencing by microinjection

The Southern cattle tick, Rhipicephalus (Boophilus) microplus, vectors bovine babesiosis and anaplasmosis, and was eradicated from the United States over several decades by the Cattle Fever Tick Eradication Program (CFTEP); however, R. microplus is endemic in Mexico and remains a continuing threat to U.S. cattle production.  Widespread and multiple acaricide resistance threatens continued success of the CFTEP to prevent and eradicate outbreak tick infestations within the United States, strongly indicating a need to develop new control methods.  Gene silencing is a valuable tool for functional genomics and validation of targets for control intervention.  Use of multiple, small ds-RNA constructs targeting the same gene is recommended to control for off-target effects that can compromise evaluation of gene silencing results, however gene silencing in ticks is a lengthy, laborious, and low-throughput process, commonly utilizing microinjection of double-stranded RNA into ticks for silencing of target transcripts.  We report construction of a dual luciferase reporter system for evaluation of gene silencing efficacy in R. microplus cell culture.  In addition, we report validation of the reporter system by comparing gene silencing efficacy obtained in vitro to quantitative reverse transcription-PCR (qRT-PCR) and phenotypic parameters obtained after in vivo microinjection of the dsRNA constructs for three different R. microplus genes.  The dual luciferase system greatly increases throughput efficiency, reduces time to obtain comparative silencing efficacy of small dsRNA constructs at greatly reduced cost, thereby enabling selection of multiple efficacious dsRNA constructs prior to final in vivo studies. 

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