Calcium ions as efficient cofactor of polycation-mediated gene transfer.
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.
Haberland
A
A
Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, Wiltberg Strasse 50, D-13122, Berlin-Buch, Germany.
Knaus
T
T
Zaitsev
S V
SV
Stahn
R
R
Mistry
A R
AR
Coutelle
C
C
Haller
H
H
Böttger
M
M
D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002130:Calcium Phosphates / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D006657:Histones; D006801:Humans; D009543:Nifedipine / Q000494:pharmacology; D011073:Polyamines; D000071228:Polyelectrolytes; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D000001:Calcimycin; D002118:Calcium; D002130:Calcium Phosphates; D002413:Cations, Divalent; D004247:DNA; D006657:Histones; D009543:Nifedipine; D011073:Polyamines; D000071228:Polyelectrolytes; C024063:nuclear protein H1(0)