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

Protist ◽

10.1016/j.protis.2021.125830 ◽

2021 ◽

pp. 125830

Author(s):

Elin Einarsson ◽

Imen Lassadi ◽

Jana Zielinski ◽

Qingtian Guan ◽

Tobias Wyler ◽

...

Keyword(s):

Genetic Model ◽

Model Organism ◽

Perkinsus Marinus

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Towards the Use of a Tree Branching Logic in the Environment of a Hospital Information System

Methods of Information in Medicine ◽

10.1055/s-0038-1636658 ◽

1980 ◽

Vol 19 (04) ◽

pp. 187-194

Author(s):

J.-Ph. Berney ◽

R. Baud ◽

J.-R. Scherrer

Keyword(s):

Syntactic Structure ◽

Large Data ◽

Selection System ◽

Frame Selection ◽

Knowing That ◽

Medical Language ◽

Selection Systems ◽

Definition Of ◽

Man Machine Dialogue ◽

Oriented System

It is well known that Frame Selection Systems (FFS) have proved both popular and effective in physician-machine and patient-machine dialogue. A formal algorithm for definition of a Frame Selection System for handling man-machine dialogue is presented here. Besides, it is shown how the natural medical language can be handled using the approach of a tree branching logic. This logic appears to be based upon ordered series of selections which enclose a syntactic structure. The external specifications are discussed with regard to convenience and efficiency. Knowing that all communication between the user and the application programmes is handled only by FSS software, FSS contributes to achieving modularity and, therefore, also maintainability in a transaction-oriented system with a large data base and concurrent accesses.

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Multiple Effects of Genetic Background on Variegated Transgene Expression in Mice

Genetics ◽

10.1093/genetics/160.3.1107 ◽

2002 ◽

Vol 160 (3) ◽

pp. 1107-1112

Author(s):

Margaret L Opsahl ◽

Margaret McClenaghan ◽

Anthea Springbett ◽

Sarah Reid ◽

Richard Lathe ◽

...

Keyword(s):

Transgene Expression ◽

Milk Protein ◽

Position Effect ◽

Position Effect Variegation ◽

Parental Population ◽

Expression Levels ◽

Variable Expression ◽

Specific Effects ◽

Milk Protein Genes ◽

Β Lactoglobulin

Abstract BLG/7 transgenic mice express an ovine β-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA × C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

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Induction of diploid gynogenesis in an evolutionary model organism, the three-spined stickleback (Gasterosteus aculeatus)

BMC Developmental Biology ◽

10.1186/1471-213x-11-55 ◽

2011 ◽

Vol 11 (1) ◽

pp. 55 ◽

Cited By ~ 5

Author(s):

Irene E Samonte-Padilla ◽

Christophe Eizaguirre ◽

Jörn P Scharsack ◽

Tobias L Lenz ◽

Manfred Milinski

Keyword(s):

Gasterosteus Aculeatus ◽

Model Organism ◽

Evolutionary Model ◽

Diploid Gynogenesis

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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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An efficient selection system for packaging herpes simplex virus amplicons.

Journal of General Virology ◽

10.1099/0022-1317-79-1-125 ◽

1998 ◽

Vol 79 (1) ◽

pp. 125-131 ◽

Cited By ~ 18

Author(s):

X Zhang ◽

S Inglis ◽

M Boursnell ◽

S Efstathiou ◽

H O'Shea ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Selection System ◽

Simplex Virus ◽

Efficient Selection

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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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