In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.
The inhibition effect of serum on the transfection efficiency tion efficiency of lipoplexes by their cationic lipid:DNA of cationic liposome-DNA complexes (lipoplexes) is a ratios, types of liposomes, incubation time in polyanion major obstacle to the application of this gene delivery veccontaining media, and time of serum addition, are tor both in vitro and in vivo. The size of the lipoplexes, as mediated mainly through size. Lipoplexes at a 2:1 charge they are presented to targeted cells, is found to be the ratio grow in size in media containing polyanions. The size major determinant of their effectiveness in transfection.growth may be arrested by adding serum to the incubation The transfection efficiency and the cell association and media. By using large lipoplexes, especially those made uptake of lipoplexes with CHO cells was found to increase from multilamellar vesicles, the serum inhibition effect may with increasing lipoplex size. The influence on the transfecbe overcome.
The association between liposome-DNA complexes (lipoplexes) and targeted cell membranes is a limiting step of cationic liposome-mediated transfection. A novel technique was developed where lipoplex-cell membrane association is enhanced by the addition of 2-6% polyethylene glycol (PEG) to the transfection media. Lipoplex-cell association was found to increase up to 100 times in the presence of PEG. Transfection increased correspondingly in the presence of PEG. This increase was found in several cell lines. These results show that lipoplex adsorption to cell membranes is a critical step in liposome-mediated transfection. This step can be facilitated by PEG-induced particle aggregation.
We have demonstrated that the viability of electrodorf desktop centrifuge. Pelleting improves the cell viability transfected adherent CHO and suspended NK-L, K-562, over the whole range of the NK-L, K-562, L1210 and MC2 L1210 and MC2 cells is improved if pelleting by centrifugcell concentrations studied. When this pelleting method is ation is performed immediately after pulsing. The protecapplied to load CHO cells with FITC-dextran (41 000 MW), tion effect on cell viability is cell line-and pellet thicknessnot only is the success rate close to 100%, but the growth dependent. For forming CHO cell pellets, centrifugation rate is similar to the control, which is far better than the force (300-13 000 g) and duration are not crucial; about conventional electroporation method. Furthermore, the five to 10 cell layers in the pellet provide the optimal protectransfection efficiency of the five cell lines in pellet is sigtion effect. NK-L, K-562, L1210 and MC2 cell pellets are nificantly higher than that in suspension. optimally formed by centrifugation at 13 000 g in an Eppen-
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.