Intracellular delivery of proteins into mouse Müller glia cells in vitro and in vivo using Pep-1 transfection reagent.
J Neurosci Methods
; 177(2): 403-19, 2009 Mar 15.
Article
em En
| MEDLINE
| ID: mdl-19056421
ABSTRACT
Direct protein transfection is a potentially valuable tool for studying protein function in basic and clinical research. A major challenge is to enable a sufficiently large amount of protein to penetrate the plasma membrane of the transfected cells. Pep-1, a protein transfection reagent, was evaluated for its ability and efficiency in delivering proteins and antibodies into mouse Müller cells in vitro and in vivo. Pep-1 delivered active beta-galactosidase enzyme and antibodies (non-specific IgG and Cy3-conjugated anti-vimentin) into cultured Müller cells with high efficiency. Transfection efficiency increased with increasing concentration of the protein in the complex and with incubation time. Following intravitreal injection of Pep-1/IgG complexes in vivo, retinal histology was preserved and immunostaining showed that the antibodies were distributed widely across the retinal surface, with the most intense staining located near the retino-vitreal border. For complexes using non-specific IgG, double staining with anti-glutamine synthetase identified many IgG-positive cells as Müller glia. IgG immunoreactivity was also detected in the cytoplasm and occasionally in the nuclei of inner retinal neurons. Dark-adapted flash electroretinogram (ERG) recordings from injected eyes were nearly identical to ERG recordings from control eyes, suggesting that injection of Pep-1/IgG complex has minimal effects on retinal function. Therefore, Pep-1 is a useful tool for intracellular delivery of antibodies to study the role of proteins in living cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
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Retina
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Transfecção
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Proteínas
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Neuroglia
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Sistemas de Liberação de Medicamentos
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Cisteamina
Limite:
Animals
Idioma:
En
Revista:
J Neurosci Methods
Ano de publicação:
2009
Tipo de documento:
Article