Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae
Open Access
- 21 January 2020
- journal article
- research article
- Published by MDPI AG in International Journal of Molecular Sciences
- Vol. 21 (3), 718
- https://doi.org/10.3390/ijms21030718
Abstract
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.Funding Information
- Interreg (0055 ALGARED PLUS 5)
This publication has 54 references indexed in Scilit:
- Microalgae for Biofuels and Animal FeedsEnergies, 2013
- Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applicationsBMC Biotechnology, 2013
- Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtiiBiotechnology Progress, 2013
- Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A PeptidePLOS ONE, 2012
- Strategies for high-level recombinant protein expression in transgenic microalgae: A reviewBiotechnology Advances, 2010
- Factors effecting expression of vaccines in microalgaeBiologicals, 2009
- Carotenoid content in mutants of the chlorophyte Chlamydomonas reinhardtii with low expression levels of phytoene desaturaseProcess Biochemistry, 2008
- A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtiiCurrent Genetics, 2008
- Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vectorNature Biotechnology, 2004
- A new mathematical model for relative quantification in real-time RT-PCRNucleic Acids Research, 2001