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Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: A comparative study

TitleAgrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: A comparative study
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication2018
AuthorsMini, P., Demurtas Olivia Costantina, Valentini S., Pallara Patrizia, Aprea Giuseppe, Ferrante Paola, and Giuliano Giovanni
JournalBMC Biotechnology
Volume18
Pagination1–12
KeywordsAgrobacterium, Agrobacterium mediated transformations, article, Bacterial, bacterial DNA, bacterial strain, bacterial virulence, Cell Wall, Chlamydomonas, Chlamydomonas reinhardtii, coculture, comparative study, controlled study, DNA, electroporation, exon, Gene expression, gene expression regulation, gene insertion, gene locus, gene vector, Genes, Genetic, genetic marker, Genetic Markers, genetic transformation, Genetic Vectors, Genetically Modified, genetics, Genome, Insertional mutagenesis, luciferase, Luciferases, marker gene, Metadata, Mutagenesis, next generation sequencing, Next-generation sequencing, nonhuman, paromomycin, Plant, plant genome, Plants, procedures, Renilla, Renilla luciferin 2 monooxygenase, Reporter, reporter gene, Silencing, T-DNA, Transformation, transgene, Transgenes, transgenic plant, untranslated region
Abstract

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 20cycles 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. © 2018 The Author(s).

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URLhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85042162227&doi=10.1186%2fs12896-018-0416-3&partnerID=40&md5=0f270106213fde22ce1a7665a5d682f4
DOI10.1186/s12896-018-0416-3
Citation KeyMini2018