mRNA imaging in the chloroplast of Chlamydomonas reinhardtii using the light-up aptamer Spinach.

Autor: Guzmán-Zapata D; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico., Domínguez-Anaya Y; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico., Macedo-Osorio KS; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico., Tovar-Aguilar A; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico., Castrejón-Flores JL; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico., Durán-Figueroa NV; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico. Electronic address: nduranf@ipn.mx., Badillo-Corona JA; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología, Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 México City, Mexico. Electronic address: jbadilloc@ipn.mx.
Jazyk: angličtina
Zdroj: Journal of biotechnology [J Biotechnol] 2017 Jun 10; Vol. 251, pp. 186-188. Date of Electronic Publication: 2017 Mar 28.
DOI: 10.1016/j.jbiotec.2017.03.028
Abstrakt: Light-up aptamers are practical tools to image RNA localization in vivo. A now classical light-up aptamer system is the combination of the 3,5-difluoro-4-hydroxybenzylidene (DFHBI) fluorogen and the RNA aptamer Spinach, which has been successfully used in bacterial and mammalian cells. However, light-up aptamers have not been used in algae. Here, we show that a simple vector, carrying Spinach, transcriptionally fused to the aphA-6 gene, can be effectively used to generate a functional light-up aptamer in the chloroplast of Chlamydomonas reinhardtii. After incubation with DFHBI, lines expressing the aphA-6/Spinach mRNA were observed with laser confocal microscopy to evaluate the functionality of the light-up aptamer in the chloroplast of C. reinhardtii. Clear and strong fluorescence was localized to the chloroplast, in the form of discrete spots. There was no background fluorescence in the strain lacking Spinach. Light-up aptamers could be further engineered to image RNA or to develop genetically encoded biosensors in algae.
(Copyright © 2017 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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