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TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation.

ABSTRACT: BACKGROUND:Transformation of microalgae to obtain recombinant proteins, lipids or metabolites of economic value is of growing interest due to low costs associated with culture growth and scaling up. At present there are only three stable nuclear selection markers for the transformation of Chlamydomonas reinhardtii, which is the most commonly transformed microalgae, specifically: the aminoglycoside phosphotransferaseses aph7and aphVIII and the phleomycin resistance ble gene. As several microalgae are resistant to some of the antibiotics associated with the mentioned resistance genes, we have developed another alternative, tetX, a NADP-requiring Oxidoreductase that hydroxylates tetracycline substrates. We provide evidence that tetX can be used to obtain nuclear transformants of Chlamydomonas reinhardtii. RESULTS:We obtained nuclear transformants harbouring the tetX gene under the control of beta 2 tubulin or HSP70ARBCS2 promoters at an efficiency of transformation of 3.28 and 6.18 colony forming units/?g DNA respectively. This is the first report of a eukaryotic cell transformed using tetracycline as a selectable marker. CONCLUSIONS:We developed a protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100 ?g/mL. We believe tetX can be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic.

SUBMITTER: Garcia-Echauri SA

PROVIDER: S-EPMC4407551 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation.

Garcia-Echauri Sergio A SA Cardineau Guy A GA

Plant methods 20150415

<h4>Background</h4>Transformation of microalgae to obtain recombinant proteins, lipids or metabolites of economic value is of growing interest due to low costs associated with culture growth and scaling up. At present there are only three stable nuclear selection markers for the transformation of Chlamydomonas reinhardtii, which is the most commonly transformed microalgae, specifically: the aminoglycoside phosphotransferaseses aph7and aphVIII and the phleomycin resistance ble gene. As several mi ...[more]

PMID: 25908936

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Project description:BACKGROUND:Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. RESULTS:We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci. CONCLUSIONS:Due to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed.

Dataset Information

TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation.

Publications

TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation.

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