Quantitative analysis of correlation between number of nuclear plasmids and gene expression activity after transfection with cationic liposomes.
OBJECTIVE
A quantitative understanding of the intracellular trafficking of plasmids delivered by nonviral vectors is essential for optimizing vector functions to increase their transfection efficiency. In this study, quantitative methods were developed to measure plasmids delivered to the nucleus, and the relationship between transfection activity and the number of plasmids in the nucleus were analyzed.


METHODS
AH130 cells were transfected with plasmids in cationic liposomes at various doses. The nuclear fraction was isolated after NP-40 lysis. and the unincorporated plasmids were enzvmatically degraded and washed away. Intranuclear plasmids were amplified by quantitative PCR. and the number of plasmids was determined. Plasmid amounts in the nucleus were also measured by Southern analysis to confirm the quantification.


RESULTS
Both methods led to similar results in measuring the nuclear plasmids within the same order of magnitude. A remarkable saturation was found for transfection activity vs. number of plasmids in the nucleus, whereas no saturation was observed in nuclear-delivered plasmids vs. dose.


CONCLUSIONS
These results clearly demonstrate the importance of the quantitative measurement of intracellular trafficking of plasmids after transfection. The findings herein described suggest that efficient transgene expression as well as enhanced nuclear delivery is required in order to achieve the maximal transfection activity of nonviral vectors.
Tachibana
Rieko
R
Faculty of Pharmaceutical Sciences, The University of Tokushima, Japan.
Harashima
Hideyoshi
H
Ide
Naoko
N
Ukitsu
Sachiko
S
Ohta
Yasuko
Y
Suzuki
Norio
N
Kikuchi
Hiroshi
H
Shinohara
Yasuo
Y
Kiwada
Hiroshi
H
D000818:Animals; D002412:Cations; D002467:Cell Nucleus / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D008081:Liposomes; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D002412:Cations; D008081:Liposomes