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      Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum

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          Abstract

          The social amoeba Dictyostelium discoideum is a model for a wide range of biological processes including chemotaxis, cell-cell communication, phagocytosis, and development. Interrogating these processes with modern genetic tools often requires the expression of multiple transgenes. While it is possible to transfect multiple transcriptional units, the use of separate promoters and terminators for each gene leads to large plasmid sizes and possible interference between units. In many eukaryotic systems this challenge has been addressed through polycistronic expression mediated by 2A viral peptides, permitting efficient, co-regulated gene expression. Here, we screen the most commonly used 2A peptides, porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A), for activity in D. discoideum and find that all the screened 2A sequences are effective. However, combining the coding sequences of two proteins into a single transcript leads to notable strain-dependent decreases in expression level, suggesting additional factors regulate gene expression in D. discoideum that merit further investigation. Our results show that P2A is the optimal sequence for polycistronic expression in D. discoideum, opening up new possibilities for genetic engineering in this model system.

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          Most cited references53

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          The genome of the social amoeba Dictyostelium discoideum.

          The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.
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            Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.

            Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.
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              mScarlet: a bright monomeric red fluorescent protein for cellular imaging

              An extremely bright, truly monomeric RFP, mScarlet, is described that outperforms existing RFPs in diverse labeling applications, especially in FRET with ratiometric imaging.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLOS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                2023
                2 March 2023
                : 18
                : 3
                : e0281211
                Affiliations
                [1 ] Department of Biomedical Engineering, Boston University, Boston, MA, United States of America
                [2 ] Biological Design Center, Boston University, Boston, MA, United States of America
                Consejo Superior de Investigaciones Cientificas, SPAIN
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States of America

                Author information
                https://orcid.org/0000-0002-4385-4060
                https://orcid.org/0000-0002-6549-1838
                https://orcid.org/0000-0001-7873-2779
                https://orcid.org/0000-0003-2155-3918
                Article
                PONE-D-22-08221
                10.1371/journal.pone.0281211
                9980757
                36862626
                4141ea55-78a0-43b3-b4f3-6c16f57fd965
                © 2023 Zhu et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 19 March 2022
                : 18 January 2023
                Page count
                Figures: 4, Tables: 1, Pages: 20
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000152, Division of Molecular and Cellular Biosciences;
                Award ID: MCB-1838341
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000861, Burroughs Wellcome Fund;
                Award ID: Career Award at the Scientific Interface
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: R35 GM133616
                Award Recipient :
                Funded by: Fonds de Recherche du Québec - Nature et technologies (FRQNT)
                Award ID: Doctoral Scholarship
                Award Recipient :
                Funded by: Boston University
                Award ID: Biological Design Center Microbiome Initiative Fellowship Program
                Award Recipient :
                Funded by: Boston University’s Rajen Kilachand Fund for Integrated Life Sciences and Engineering
                Award ID: Multicellular Design Program Fellowship
                Award Recipient :
                Funded by: Life Sciences Research Foundation
                Award ID: Simons Foundation Awardee
                Award Recipient :
                "This work was supported by the National Science Foundation grant MCB-1838341 to A.E.S, the National Institutes of Health (NIGMS) grant R35 GM133616 to A.E.S., and the Burroughs Wellcome Fund Career Award at the Scientific Interface to A.E.S. X.Z. was partially supported by the Fonds de recherche du Québec - Nature et technologies (FRQNT). C.R.T. was partially supported by the Biological Design Center Microbiome Initiative Fellowship Program. E.R.H. was partially supported by a Multicellular Design Program fellowship from Boston University’s Rajen Kilachand Fund for Integrated Life Sciences and Engineering and is a Simons Foundation Awardee of the Life Sciences Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."
                Categories
                Research Article
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Dictyostelium Discoideum
                Research and Analysis Methods
                Model Organisms
                Dictyostelium Discoideum
                Biology and Life Sciences
                Organisms
                Eukaryota
                Protists
                Slime Molds
                Dictyosteliomycota
                Dictyostelium
                Dictyostelium Discoideum
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Protozoan Models
                Dictyostelium
                Dictyostelium Discoideum
                Research and Analysis Methods
                Spectrum Analysis Techniques
                Spectrophotometry
                Cytophotometry
                Flow Cytometry
                Biology and life sciences
                Genetics
                Gene expression
                DNA transcription
                Biology and Life Sciences
                Genetics
                Gene Expression
                Viral Gene Expression
                Biology and Life Sciences
                Genetics
                Microbial Genetics
                Viral Genetics
                Viral Gene Expression
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Genetics
                Viral Gene Expression
                Biology and Life Sciences
                Microbiology
                Microbial Control
                Antimicrobial Resistance
                Antibiotic Resistance
                Medicine and Health Sciences
                Pharmacology
                Antimicrobial Resistance
                Antibiotic Resistance
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Research and analysis methods
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Transcription Factors
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Transcription Factors
                Biology and Life Sciences
                Biochemistry
                Proteins
                Regulatory Proteins
                Transcription Factors
                Custom metadata
                Data and links to the Python code for analysis and re-creating the figures are available on a Dryad repository (DOI: 10.5061/dryad.c866t1g8r). Plasmids for the 2A sequences and the codon-optimized fluorescent proteins created for this study are available from the Dicty Stock Center at http://dictybase.org/StockCenter/StockCenter.html.

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