Development of the Myzozoan Aquatic Parasite Perkinsus marinus as a Versatile Experimental Genetic Model Organism

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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Saccharomyces cerevisiae (Baker’s Yeast) as an Interfering RNA Expression and Delivery System

Current Drug Targets ◽

10.2174/1389450120666181126123538 ◽

2019 ◽

Vol 20 (9) ◽

pp. 942-952 ◽

Cited By ~ 9

Author(s):

Molly Duman-Scheel

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Interference ◽

Genetic Model ◽

Model Organism ◽

Rna Expression ◽

Commercial Development ◽

Rna Integrity ◽

Yeast Cultures ◽

Ideal System ◽

Interfering Rna

The broad application of RNA interference for disease prevention is dependent upon the production of dsRNA in an economically feasible, scalable, and sustainable fashion, as well as the identification of safe and effective methods for RNA delivery. Current research has sparked interest in the use of Saccharomyces cerevisiae for these applications. This review examines the potential for commercial development of yeast interfering RNA expression and delivery systems. S. cerevisiae is a genetic model organism that lacks a functional RNA interference system, which may make it an ideal system for expression and accumulation of high levels of recombinant interfering RNA. Moreover, recent studies in a variety of eukaryotic species suggest that this microbe may be an excellent and safe system for interfering RNA delivery. Key areas for further research and development include optimization of interfering RNA expression in S. cerevisiae, industrial-sized scaling of recombinant yeast cultures in which interfering RNA molecules are expressed, the development of methods for largescale drying of yeast that preserve interfering RNA integrity, and identification of encapsulating agents that promote yeast stability in various environmental conditions. The genetic tractability of S. cerevisiae and a long history of using this microbe in both the food and pharmaceutical industry will facilitate further development of this promising new technology, which has many potential applications of medical importance.

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Alzheimer’s disease-relevant tau modifications selectively impact neurodegeneration and mitophagy in a novel C. elegans single-copy transgenic model

10.1101/2020.02.12.946004 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sanjib Guha ◽

Sarah Fischer ◽

Gail VW Johnson ◽

Keith Nehrke

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Genetic Model ◽

Neurodegenerative Disorder ◽

Model Organism ◽

Single Copy ◽

Mitochondrial Stress ◽

C Elegans ◽

Touch Receptor Neurons ◽

New Perspective

ABSTRACTBackgroundA defining pathological hallmark of the progressive neurodegenerative disorder Alzheimer’s disease (AD) is the accumulation of misfolded tau with abnormal post-translational modifications (PTMs). These include phosphorylation at Threonine 231 (T231) and acetylation at Lysine 274 (K274) and at Lysine 281 (K281). Although tau is recognized to play a central role in pathogenesis of AD, the precise mechanisms by which these abnormal PTMs contribute to the neural toxicity of tau is unclear.MethodsHuman 0N4R tau (wild type) was expressed in touch receptor neurons of the genetic model organism C. elegans through single-copy gene insertion. Defined mutations were then introduced into the single-copy tau transgene through CRISPR-Cas9 genome editing. These mutations included T231E and T231A, to mimic phosphorylation and phospho-ablation of a commonly observed pathological epitope, respectively, and K274/281Q, to mimic disease-associated lysine acetylation. Stereotypical touch response assays were used to assess behavioral defects in the transgenic strains as a function of age, and genetically-encoded fluorescent biosensors were used to measure the morphological dynamics and turnover of touch neuron mitochondria.ResultsUnlike existing tau overexpression models, C. elegans single-copy expression of tau did not elicit overt pathological phenotypes at baseline. However, strains expressing disease associated PTM-mimetics (T231E and K274/281Q) exhibited reduced touch sensation and morphological abnormalities that increased with age. In addition, the PTM-mimetic mutants lacked the ability to engage mitophagy in response to mitochondrial stress.ConclusionsLimiting the expression of tau results in a genetic model where pathological modifications and age result in evolving phenotypes, which may more closely resemble the normal progression of AD. The finding that disease-associated PTMs suppress compensatory responses to mitochondrial stress provides a new perspective into the pathogenic mechanisms underlying AD.

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Creating Porcine Biomedical Models Through Recombineering

Comparative and Functional Genomics ◽

10.1002/cfg.404 ◽

2004 ◽

Vol 5 (3) ◽

pp. 262-267 ◽

Cited By ~ 7

Author(s):

Margarita M. Rogatcheva ◽

Laurie A. Rund ◽

Kelly S. Swanson ◽

Brandy M. Marron ◽

Jonathan E. Beever ◽

...

Keyword(s):

Genetic Information ◽

Positional Cloning ◽

Reverse Genetics ◽

Genetic Model ◽

Phenotypic Diversity ◽

Model Organism ◽

Artificial Chromosome ◽

Forward Genetics ◽

Working Definition ◽

Definition Of

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide ‘clones’ of genetically modified animals.

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Reproductive fitness and dietary choice behavior of the genetic model organism Caenorhabditis elegans under semi-natural conditions

Molecules and Cells ◽

10.1007/s10059-010-0125-9 ◽

2010 ◽

Vol 30 (4) ◽

pp. 347-353 ◽

Cited By ~ 12

Author(s):

Katharina Freyth ◽

Tim Janowitz ◽

Frank Nunes ◽

Melanie Voss ◽

Alexander Heinick ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Choice Behavior ◽

Genetic Model ◽

Model Organism ◽

Reproductive Fitness ◽

Natural Conditions ◽

Dietary Choice

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Saccharomyces cerevisiae as a Genetic Model Organism

Genetic Techniques for Biological Research ◽

10.1002/0470846623.ch1 ◽

2003 ◽

pp. 1-22

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Model ◽

Model Organism

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Quantifying Tubulin Concentration and Microtubule Number Throughout the Fission Yeast Cell Cycle

Biomolecules ◽

10.3390/biom9030086 ◽

2019 ◽

Vol 9 (3) ◽

pp. 86 ◽

Cited By ~ 4

Author(s):

Isabelle Loiodice ◽

Marcel Janson ◽

Penny Tavormina ◽

Sebastien Schaub ◽

Divya Bhatt ◽

...

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Genetic Model ◽

Spindle Pole Body ◽

Model Organism ◽

Spindle Pole ◽

Living Cells ◽

Fluorescent Imaging ◽

Mother And Daughter ◽

In The Wild

The fission yeast Schizosaccharomyces pombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αβ-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αβ-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

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The manifold structure of limb coordination in walking Drosophila

eLife ◽

10.7554/elife.46409 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 24

Author(s):

Brian D DeAngelis ◽

Jacob A Zavatone-Veth ◽

Damon A Clark

Keyword(s):

Genetic Model ◽

Model Organism ◽

Legged Locomotion ◽

Neural Activation ◽

Symmetric Pattern ◽

Multiple Dimensions ◽

Limb Coordination ◽

Spontaneous Behavior ◽

Low Dimensional ◽

Coordination Patterns

Terrestrial locomotion requires animals to coordinate their limb movements to efficiently traverse their environment. While previous studies in hexapods have reported that limb coordination patterns can vary substantially, the structure of this variability is not yet well understood. Here, we characterized the symmetric and asymmetric components of variation in walking kinematics in the genetic model organism Drosophila. We found that Drosophila use a single continuum of coordination patterns without evidence for preferred configurations. Spontaneous symmetric variability was associated with modulation of a single control parameter—stance duration—while asymmetric variability consisted of small, limb-specific modulations along multiple dimensions of the underlying symmetric pattern. Commands that modulated walking speed, originating from artificial neural activation or from the visual system, evoked modulations consistent with spontaneous behavior. Our findings suggest that Drosophila employ a low-dimensional control architecture, which provides a framework for understanding the neural circuits that regulate hexapod legged locomotion.

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Calcineurin homologous proteins regulate the membrane localization and activity of sodium/proton exchangers in C. elegans

AJP Cell Physiology ◽

10.1152/ajpcell.00291.2015 ◽

2016 ◽

Vol 310 (3) ◽

pp. C233-C242 ◽

Cited By ~ 4

Author(s):

Erik Allman ◽

Qian Wang ◽

Rachel L. Walker ◽

Molly Austen ◽

Maureen A. Peters ◽

...

Keyword(s):

Genetic Model ◽

Expression Patterns ◽

Critical Role ◽

Model Organism ◽

Loss Of Function ◽

Homologous Proteins ◽

Relative Significance ◽

C Elegans ◽

Fluorescent Tags

Calcineurin B homologous proteins (CHP) are N-myristoylated, EF-hand Ca2+-binding proteins that bind to and regulate Na+/H+ exchangers, which occurs through a variety of mechanisms whose relative significance is incompletely understood. Like mammals, Caenorhabditis elegans has three CHP paralogs, but unlike mammals, worms can survive CHP loss-of-function. However, mutants for the CHP ortholog PBO-1 are unfit, and PBO-1 has been shown to be required for proton signaling by the basolateral Na+/H+ exchanger NHX-7 and for proton-coupled intestinal nutrient uptake by the apical Na+/H+ exchanger NHX-2. Here, we have used this genetic model organism to interrogate PBO-1's mechanism of action. Using fluorescent tags to monitor Na+/H+ exchanger trafficking and localization, we found that loss of either PBO-1 binding or activity caused NHX-7 to accumulate in late endosomes/lysosomes. In contrast, NHX-2 was stabilized at the apical membrane by a nonfunctional PBO-1 protein and was only internalized following its complete loss. Additionally, two pbo-1 paralogs were identified, and their expression patterns were analyzed. One of these contributed to the function of the excretory cell, which acts like a kidney in worms, establishing an alternative model for testing the role of this protein in membrane transporter trafficking and regulation. These results lead us to conclude that the role of CHP in Na+/H+ exchanger regulation differs between apical and basolateral transporters. This further emphasizes the importance of proper targeting of Na+/H+ exchangers and the critical role of CHP family proteins in this process.

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DrosophilaModels of Tauopathies: What Have We Learned?

International Journal of Alzheimer s Disease ◽

10.1155/2012/970980 ◽

2012 ◽

Vol 2012 ◽

pp. 1-14 ◽

Cited By ~ 7

Author(s):

Marc Gistelinck ◽

Jean-Charles Lambert ◽

Patrick Callaerts ◽

Bart Dermaut ◽

Pierre Dourlen

Keyword(s):

Frontotemporal Dementia ◽

High Throughput ◽

Neurodegenerative Disorders ◽

Genetic Model ◽

Model Organism ◽

Molecular Pathways ◽

Protein Tau ◽

Microtubule Associated Protein Tau ◽

The Past ◽

Genome Wide

Aggregates of the microtubule-associated protein Tau are neuropathological hallmark lesions in Alzheimer's disease (AD) and related primary tauopathies. In addition, Tau is genetically implicated in a number of human neurodegenerative disorders including frontotemporal dementia (FTD) and Parkinson's disease (PD). The exact mechanism by which Tau exerts its neurotoxicity is incompletely understood. Here, we give an overview of how studies using the genetic model organismDrosophilaover the past decade have contributed to the molecular understanding of Tau neurotoxicity. We compare the different available readouts for Tau neurotoxicity in flies and review the molecular pathways in which Tau has been implicated. Finally, we emphasize that the integration of genome-wide approaches in human or mice with high-throughput genetic validation inDrosophilais a fruitful approach.

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