Induction of protein aggregation in zebrafish embryos as a method for the screening of new drugs or mutations against proteinopathies
Autor: | Octavio Monasterio, Gissela Araya, Luis Pouchucq, Pablo Lobos-Ruiz |
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Rok vydání: | 2018 |
Předmět: |
0301 basic medicine
Protein aggregate induction Clinical Biochemistry Protein aggregation medicine.disease_cause 03 medical and health sciences In vivo Biochemistry Genetics and Molecular Biology Fluorescence microscope medicine Aggregate size distribution lcsh:Science Zebrafish ComputingMethodologies_COMPUTERGRAPHICS Mutant screening Mutation biology Drug discovery Chemistry Intracellular protein aggregation biology.organism_classification Cell biology Induction of protein aggregates in zebrafish early embryos by direct microinjection of denatured protein Medical Laboratory Technology 030104 developmental biology Tubulin biology.protein lcsh:Q Ultracentrifuge Zebrafish embryo Intracellular |
Zdroj: | MethodsX, Vol 5, Iss, Pp 322-327 (2018) MethodsX |
ISSN: | 2215-0161 |
Popis: | Graphical abstract The sustained increase in the prevalence of protein aggregation related diseases requires the development of feasible methods for the design of therapeutic alternatives. The procedure traditionally used for the search of drugs or therapeutic mutations includes in vitro experiments, designed to prevent the aggregation of model proteins, which are then complemented with cellular toxicity studies in vivo, slowing down the finding of solutions. To address this, we have developed a protocol that facilitates the search of molecules and anti-aggregation mutations since it allows to evaluate their therapeutic capabilities directly in in vivo experiments with the use of zebrafish early embryos. Avoiding the necessity of performing in vitro and in vivo procedures separately. Giving a more realistic method for the results interpretation. Zebrafish embryos were induced to produce intracellular aggregates of proteins by simple microinjections of known quantities of an aggregation prone protein previously labeled. The toxicity was evaluated by the survival of the embryos, while the formation of aggregates was quantified by fluorescence microscopy. The size distribution of the protein aggregates was revealed by means of ultracentrifuge sedimentation analysis. For the development of the present method, the human γ-tubulin protein was used as model protein, which generated intracellular aggregates in more than 60% of the injected embryos. To evaluate the method, a mutation was performed that altered the state of intracellular aggregation of γ-tubulin, obtaining a significant decrease in the amount and size of the intracellular aggregates. The present method can be used for any suitable intracellular aggregation protein model. The current method present important advantages such as: Easy induction of intracellular aggregates. Simple detection of intracellular protein aggregates through fluorescence microscopy and subcellular fractionation. Overall view of the effect of drugs or mutations by combining the toxicity, the development behavior and the size distribution of intracellular protein aggregates. |
Databáze: | OpenAIRE |
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