lipofectamine and dioctadecylamidoglycylspermine

A monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 microm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINE), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.

Topics: Animals; Cation Exchange Resins; Cations; Cattle; Cell Line; Cricetinae; Culture Media; Evaluation Studies as Topic; Fatty Acids, Monounsaturated; Gene Expression; Gene Transfer Techniques; Glycine; Humans; Indicators and Reagents; Lipids; Luciferases; Particle Size; Phosphatidylethanolamines; Plasmids; Quaternary Ammonium Compounds; Spermine; Transfection

Cationic liposomes are an alternative non-viral vector for gene therapy, but several factors affect transfection efficiency. A novel cationic lipid, o-ethyldioleoylphosphatidylcholinium (EDOPC), was studied for characterization of the time course and effects of lipid composition, concentration, charge ratio, mixing techniques, passage number, and stimulated state on transfection of human vascular cells, represented by human umbilical vein endothelial cells (HUVEC).. HUVEC cultures were seeded at a density of 45,000 cells/well in 24-well plates and incubated overnight. Triplicate wells were transfected with samples of EDOPC/reporter plasmid for 2 h, followed by a 24-h expression time, which was the peak expression time point in an initial time-course experiment. Measuring luciferase in cell lysates quantitated gene expression.. Transfection of HUVEC with EDOPC was optimal with a concentration of 100 microgram lipid/well, ratio of 3:1 EDOPC:plasmid, fractional mixing of lipid and plasmid, centrifugation, and incubation in serum-free media. Transfections in sequential passages showed striking decreases in gene expression and regression analysis revealed the relationship: RLU = 120,000 - (10, 400 x passage number), r(2) = 0.947. HUVEC activated by cytokine stimulation remain susceptible to gene transfer specifically with EDOPC.. During transfection of HUVEC with cationic lipid species, an increase in passage number is associated with linear reduction in luciferase expression, and hence passage number must be controlled in comparative experiments. Characteristics of EDOPC may permit site-specific efficient transfection of activated human vascular cells that can be isolated from serum by mechanical methods.

Topics: Blood Proteins; Cation Exchange Resins; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Drug Carriers; Endothelium, Vascular; Gene Expression; Gene Transfer Techniques; Genes, Reporter; Glycine; Humans; Lipid Metabolism; Lipids; Luciferases; Oleic Acids; Phosphatidylcholines; Plasmids; Recombinant Proteins; Spermine; Transfection; Tumor Necrosis Factor-alpha

We have examined lipids as transfection agents to introduce recombinant plasmids into primary cultures of rat hippocampal neurons. By modifying the protocol for transfection mediated by the commercial reagent DOTAP, we were able to achieve a transfection efficiency of about 3%. Expression of various transfected gene products was sustained for several weeks in culture, the neurons developed normally and the transfected gene products were targeted to the appropriate subcellular compartment.

Topics: Animals; Cation Exchange Resins; Cells, Cultured; DNA; Drug Carriers; Fatty Acids, Monounsaturated; Fluorescent Antibody Technique; Fluorescent Dyes; Glycine; Hippocampus; Immunoglobulin kappa-Chains; Lipids; Mice; Neurons; Plasmids; Quaternary Ammonium Compounds; Rats; Recombinant Proteins; Spermine; Transfection

Direct in vivo transfection of tumor nodules in situ via liposome-DNA complexes has been employed as a strategy to accomplish antitumor immunization. To circumvent the potential safety hazards associated with systemic localization of delivered DNA, the utility of mRNA transcript-mediated gene delivery was explored. Capped, polyadenylated mRNA transcripts encoding the firefly luciferase and Escherichia coli lacZ reporter genes were derived by in vitro transcription. Transfection of the human breast cancer cell line MDA-MB-435 in vitro was accomplished employing cationic liposome-mRNA complexes. Evaluation of a panel of cationic liposome preparations demonstrated significant differences in the capacity of the various preparations to accomplish mRNA-mediated transfection. Quantitative evaluation of in vitro transfection demonstrated that target cells could be transfected at a high level of efficiency. The mRNA liposome-complexes were evaluated for in vivo transfection of tumor nodules in human xenografts in athymic nude mice. It could be demonstrated the liposome-mRNA complexes were comparable in efficacy to liposome-DNA complexes in accomplishing in situ tumor transfection. Thus, mRNA may be considered as an alternative to plasmid DNA as a gene transfer vector for genetic immunopotentiation applications.

Topics: beta-Galactosidase; Breast Neoplasms; Cation Exchange Resins; Cations; Drug Carriers; Fatty Acids, Monounsaturated; Genes, Reporter; Genetic Therapy; Glycine; Humans; Lipids; Liposomes; Luciferases; Phosphatidylethanolamines; Quaternary Ammonium Compounds; Recombinant Fusion Proteins; RNA, Messenger; Safety; Spermine; Transfection; Tumor Cells, Cultured