Development of the myzozoan aquatic parasite Perkinsus marinus as a versatile experimental genetic model organism
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Peer-reviewed
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Authors
Lassadi, Imen
Zielinski, Jana
Guan, Qingtian
Wyler, Tobias
Abstract
ABSTRACT
The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to, and share much of their biology with, dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation has been previously achieved for select Perkinsus spp. but with few tools for transgene expression and only limited selection efficacy. We thus sought to expand the power of experimental genetic tools for Perkinsus marinus — the principal perkinsozoan model to date. We constructed a modular plasmid assembly system that enables expression of multiple genes simultaneously. We developed an efficient selection system for three drugs, puromycin, bleomycin and blasticidin, that achieves transformed cell populations in as little as three weeks. We developed and quantified eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination and often involves transgene fragmentation including deletion of some introduced elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection systems to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide new capacity to efficiently genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.Description
Keywords
31 Biological Sciences, 3105 Genetics, Biotechnology, Genetics, Generic health relevance
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1434-4610
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Elsevier
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We are grateful to Mark Carrington for providing the BiP
antibody, Eelco Tromer for technical assistance with codonoptimisation of construct parts, Konstantin Barylyuk and
Tom Smith for assistance with statistical analyses, and
Febrimarsa for introducing Perkinsus transformation to our
studies. This work was supported by grants from the
Gordon and Betty Moore Foundation (GBMF 4977,
4977.01) and King Abdullah University of Science and
Technology (KAUST) (BAS/1/1020-01-01)