期刊
MOLECULAR AND BIOCHEMICAL PARASITOLOGY
卷 195, 期 1, 页码 1-5出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.molbiopara.2014.05.002
关键词
Leishmania; Firefly luciferase; Renilla luciferase; Gene regulation; Translational regulation; Nutrient stress response
资金
- Oregon Clinical and Translational Research Institute (OCTRI) [UL1TR000128]
- National Center for Advancing Translational Sciences (NCATS)
- National Institutes of Health (NIH)
- National Institute of Allergy and Infectious Diseases [AI023682, AI044138]
Gene expression in kinetoplastid parasites is regulated via post-transcriptional mechanisms that modulate mRNA turnover, translation rate, and/or post-translational protein stability. To facilitate the analysis of post-transcriptional regulation, a dual luciferase system was developed in which firefly and Renilla luciferase reporters genetically fused to compatible drug resistance genes are integrated in place of one allele of the gene of interest and of an internal control gene, respectively, in a manner that preserves the cognate pre-mRNA processing signals. The sensitivity and reproducibility of the assay coupled with the ability to rapidly assemble reporter integration constructs render the dual luciferase system suitable for analysis of multiple candidates derived from global expression analysis platforms. To demonstrate the utility of the system, regulation of three genes in response to purine starvation was examined in Leishmania donovani promastigotes. This dual luciferase system should be directly applicable to the analysis of post-transcriptional regulation in other kinetoplastids. (C) 2014 Elsevier B.V. All rights reserved.
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