Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus

Cold, Emma R.; Vasta, Gerardo R.; Robledo, José A. Fernández

doi:10.1645/16-88

Cited by 7 publications

(5 citation statements)

References 29 publications

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“…The availability of axenic culture and transfection methodology, the parasite's ability to naturally trigger an immune response in mice, and phylogenetic affinities drove us to use it to express apicomplexan genes. However, these attempts were met with variable success (Wijayalath et al, 2014;Cold et al, 2017). The other laboratories often report the inconsistency of the gene expression.…”

Section: Discussionmentioning

confidence: 99%

“…The CRISPR/Cas9 method developed here can be used to understand the localization and protein-protein interactions in the parasite's life cycle and generate the transgenic parasite model. The natural adjuvant ability of the P. marinus and its potential as a novel oral vaccine platform, efforts for the expression of heterologous antigens, always relied on one plasmid p[MOE]:EK-His-GFP (Cold et al, 2017), with potential for a monovalent vaccine expression. With the availability of fast and robust CRISPR/Cas9, we can now express multiple heterologous genes and develop P. marinus as a polyvalent oral vaccine delivery system.…”

Section: Discussionmentioning

confidence: 99%

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CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Yadavalli

Umeda

Waugh

et al. 2021

Front. Bioeng. Biotechnol.

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Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Yadavalli

Umeda

Waugh

et al. 2021

Front. Bioeng. Biotechnol.

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“…To date only single coding sequences have been expressed in P. marinus, typically as fusion proteins (Cold et al, 2017;Fernández-Robledo et al, 2008;Sakamoto et al, 2016Sakamoto et al, , 2019. To allow for further development of genetic modification methods we asked if multiple genes could be expressed simultaneously from a single expression vector.…”

Section: Simultaneous Expression Of Multiple Transgenesmentioning

confidence: 99%

“…To date, all reported transformation of Perkinsus used the 5' upstream sequence of the highly expressed transcript for the MOE protein to drive protein expression (Cold et al, 2017;Fernández-Robledo et al, 2008;Sakamoto et al, 2016Sakamoto et al, , 2019Sakamoto et al, , 2021. We tested for further useful promoter sequences that might enable a range of levels of expression of transgenes.…”

Section: Expansion Of Functional Promoter Sequences and Transgene Expression Stabilitymentioning

confidence: 99%

Development of the myzozoan aquatic parasite Perkinsus marinus as a versatile experimental genetic model organism

Einarsson

Lassadi

Zielinski

et al. 2021

Preprint

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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.

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“…(Fernandez Robledo et al, 2014). This genetically tractable protozoan parasite is becoming a model system for protozoan parasites in bivalves (Cold et al, 2016;Fernandez-Robledo et al, 2008) and it has potential for medical applications (Cold et al, 2017;Wijayalath et al, 2014). Mechanistic studies at the cellular level would also benefit from cell lines and a genetically tractable system.…”

Section: Resources and Toolsmentioning

confidence: 99%

From the raw bar to the bench: Bivalves as models for human health

Robledo

Yadavalli

Allam

et al. 2019

Developmental & Comparative Immunology

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Bivalves, from raw oysters to steamed clams, are popular choices among seafood lovers and once limited to the coastal areas. The rapid growth of the aquaculture industry and improvement in the preservation and transport of seafood have enabled them to be readily available anywhere in the world. Over the years, oysters, mussels, scallops, and clams have been the focus of research for improving the production, managing resources, and investigating basic biological and ecological questions. During this decade, an impressive amount of information using high-throughput genomic, transcriptomic and proteomic technologies has been produced in various classes of the Mollusca group, and it is anticipated that basic and applied research will significantly benefit from this resource. One aspect that is also taking momentum is the use of bivalves as a model system for human health. In this review, we highlight some of the aspects of the biology of bivalves that have direct implications in human health including the shell formation, stem cells and cell

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Transient Expression of Plasmodium berghei MSP8 and HAP2 in the Marine Protozoan Parasite Perkinsus marinus

Cited by 7 publications

References 29 publications

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Development of the myzozoan aquatic parasite Perkinsus marinus as a versatile experimental genetic model organism

From the raw bar to the bench: Bivalves as models for human health

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