Calcium ions as efficient cofactor of polycation-mediated gene transfer.
Biochim Biophys Acta
; 1445(1): 21-30, 1999 Apr 14.
Article
em En
| MEDLINE
| ID: mdl-10209255
ABSTRACT
We investigated the effect of calcium on the transfection of non-viral DNA transfer systems. Cationic proteins such as the nuclear protein H1, the polycation polylysine and a number of commercial transfection agents exhibited high transfection rates in the presence of Ca2+. Without Ca2+ H1 and HMG1 were inactive in transfection of the human permanent endothelial cell line ECV 304 while cationic liposomes such as Lipofectin and Lipofectamine did not show any Ca2+ dependence. More detailed experiments showed that Ca2+ was replaceable by the lysosomotropic agent chloroquine. Furthermore, it was possible to separate the transfection-enhancing role of Ca2+ from the actual transfection process by adding Ca2+ to the cells after the transfection period and still to obtain a significant transgene expression. This makes it possible to distinguish between cellular uptake of H1 (or mediator)-DNA complexes and endocytotic release. We also replaced soluble Ca2+ by Ca-phosphate precipitates not containing DNA and obtained similar transfection results. This allowed us to suggest that the addition of free Ca2+ to the transfection medium resulted in nascent Ca-phosphate microprecipitates. The known fusogenic and membranolytic activity of such microprecipitates could facilitate the transport through and the release of the transfecting complexes from the endosomal/lysosomal compartment.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Poliaminas
/
Transfecção
/
Cálcio
Limite:
Humans
Idioma:
En
Ano de publicação:
1999
Tipo de documento:
Article