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RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene

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CONACYT-Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
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Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
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Departamento de Biotecnología, Universidad Autónoma Metropolitana, Iztapalapa 09340, Mexico
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Laboratorio de Parasitología Experimental, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
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Laboratorio de Nutrición Experimental, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
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Colegio de Ciencias y Humanidades, Plantel Casa Libertad, Universidad Autónoma de la Ciudad de México, Ciudad de México 09620, Mexico
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Laboratorio de Neurociencias, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
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Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México 04530, Mexico
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Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Genes 2017, 8(11), 303; https://doi.org/10.3390/genes8110303
Received: 14 September 2017 / Revised: 12 October 2017 / Accepted: 31 October 2017 / Published: 3 November 2017
(This article belongs to the Section Molecular Genetics and Genomics)
The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100–200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways. View Full-Text
Keywords: Giardia; NADH oxidase; drug targets; RNAi; siRNA; stem-loop RT-qPCR Giardia; NADH oxidase; drug targets; RNAi; siRNA; stem-loop RT-qPCR
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Marcial-Quino, J.; Gómez-Manzo, S.; Fierro, F.; Rufino-González, Y.; Ortega-Cuellar, D.; Sierra-Palacios, E.; Vanoye-Carlo, A.; González-Valdez, A.; Torres-Arroyo, A.; Oria-Hernández, J.; Reyes-Vivas, H. RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene. Genes 2017, 8, 303.

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