Cationic cholesterol promotes gene transfection using the nuclear localization signal in protamine.
OBJECTIVE
The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin).


METHODS
Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37 degrees C. The cells then were washed and cultured for another 40 h in the growth medium at 37 degrees C before luciferase assay.


RESULTS
The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine.


CONCLUSIONS
Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.
Graduate School of Pharmaceutical Sciences. Nagoya City University, Nagoya, Japan.
Ari
A
Noguchi
Naohide
N
Hirashima
Mamoru
M
Nakanishi
D016475:3T3 Cells; D000818:Animals; D002412:Cations / Q000008:administration & dosage / Q000737:chemistry; D002784:Cholesterol / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D019913:Nuclear Localization Signals / Q000008:administration & dosage / Q000235:genetics*; D011479:Protamines / Q000008:administration & dosage / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D002412:Cations; D019913:Nuclear Localization Signals; D011479:Protamines; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; D002784:Cholesterol