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Open AccessArticle

Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae

Laboratory of Biochemistry. Faculty of Experimental Sciences. Marine International Campus of Excellence and RENSMA. University of Huelva, 21071 Huelva, Spain
Department of Biochemistry and Molecular Biology. University of Córdoba, 14071 Córdoba, Spain
Laboratory of Biotechnology of Natural Products, Agro-feed International Excellence campus, University of Almería, 04071 Almería, Spain
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(3), 718;
Received: 19 December 2019 / Revised: 18 January 2020 / Accepted: 20 January 2020 / Published: 22 January 2020
(This article belongs to the Section Molecular Biology)
Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3′-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter. View Full-Text
Keywords: 2A; microalgae transformation; paromomycin; multicistronic transcript 2A; microalgae transformation; paromomycin; multicistronic transcript
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MDPI and ACS Style

Molina-Márquez, A.; Vila, M.; Rengel, R.; Fernández, E.; García-Maroto, F.; Vigara, J.; León, R. Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae. Int. J. Mol. Sci. 2020, 21, 718.

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