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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

Author:
A. P. Tuckow, K. B. Temeyer
Source:
Insect molecular biology 2015 v.24 no.4 pp. 454-466
ISSN:
0962-1075
Subject:
Lampyridae, RNA interference, Rhipicephalus microplus, anaplasmosis, babesiosis, cattle, cell culture, cell lines, genomics, humans, luciferase, phosphoglycerate kinase, system optimization, ticks, transcription (genetics)
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.
Agid:
62213
Handle:
10113/62213