Enhancing endosomal escape of transduced proteins by photochemical internalisation.
PLoS One
; 7(12): e52473, 2012.
Article
en En
| MEDLINE
| ID: mdl-23285056
ABSTRACT
Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Endosomas
/
Transducción Genética
/
Albúmina Sérica Bovina
/
Endocitosis
/
Procesos Fotoquímicos
Tipo de estudio:
Guideline
Límite:
Adult
/
Animals
/
Child, preschool
/
Humans
Idioma:
En
Revista:
PLoS One
Asunto de la revista:
CIENCIA
/
MEDICINA
Año:
2012
Tipo del documento:
Article
País de afiliación:
Alemania