Myoblots: dystrophin quantification by in-cell western assay for a streamlined development of Duchenne muscular dystrophy (DMD) treatments.
Neuropathol Appl Neurobiol
; 44(5): 463-473, 2018 08.
Article
en En
| MEDLINE
| ID: mdl-29086434
AIMS: New therapies for neuromuscular disorders are often mutation specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are being developed but as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate drugs are tested. Moreover, dystrophin quantification by western blotting requires a large number of cultured cells; so fewer compounds are as thoroughly screened as is desirable. We aimed to develop a quantitative assessment tool using fewer cells to contribute in the study of dystrophin and to identify better drug candidates. METHODS: An 'in-cell western' assay is a quantitative immunofluorescence assay performed in cell culture microplates that allows protein quantification directly in culture, allowing a higher number of experimental repeats and throughput. We have optimized the assay ('myoblot') to be applied to the study of differentiated myoblast cultures. RESULTS: After an exhaustive optimization of the technique to adapt it to the growth and differentiation rates of our cultures and the low intrinsic expression of our proteins of interests, our myoblot protocol allows the quantification of dystrophin and other muscle-associated proteins in muscle cell cultures. We are able to distinguish accurately between the different sets of patients based on their dystrophin expression and detect dystrophin restoration after treatment. CONCLUSIONS: We expect that this new tool to quantify muscle proteins in DMD and other muscle disorders will aid in their diagnosis and in the development of new therapies.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Western Blotting
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Distrofina
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Técnica del Anticuerpo Fluorescente
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Distrofia Muscular de Duchenne
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Mioblastos
Tipo de estudio:
Guideline
Límite:
Humans
Idioma:
En
Revista:
Neuropathol Appl Neurobiol
Año:
2018
Tipo del documento:
Article
País de afiliación:
España
Pais de publicación:
Reino Unido