DOTAP chloride

Cell lines

mammalian CV-1 and 3T3 cells

Preparation Method

Lipid dispersions containing cationic lipid analogues were prepared by first bath-sonicating dried lipid samples in distilled water to clarity (2-5 min), then adding an equal concentration of 308 mM NaCl, 40 mM Hepes (pH 7.4), and sonicating further for 2 min. Lipid/DNA mixtures for transfections were prepared by incubating dotap chloride with DNA for 5 min, typically at a total lipid concentration of 0.5-2 mg/ml, before addition to cell monolayers. Reverse-phase evaporation vesicles were prepared as described previously [2] and filtered through 0.1/xm pore-size Nucleopore filters. Lipid mixing between cationic lipid dispersions and large unilamellar PC/PS vesicles was assayed by a resonance energy transfer-based procedure as described previously [3], incubating cationic lipid dispersions (2 μM), colabeled with 1 mol% (12-CPS)-18 PC and 0.35 mol% (12-DABS)-18 PC, with a 9-fold excess (18 μM) of unlabeled PC/PS vesicles. For transfection of cells using cationic lipid dispersions, cell monolayers were washed twice with HEPESbuffered saline, then incubated with lipid/DNA mixtures in the latter medium (total volume 3 ml per100-mm culture dish) at 37 °C, for 90 min except whereotherwise indicated. Following this incubation period,the incubation medium was either replaced or supplemented with 12 ml of D-MEM containing 5% serum,and the cells were cultured for a further 36 h beforeharvesting. Chloramphenicol acetyltransferase activityin plasmid pSV2cat-transfected cells was assayed asdescribed previously [4], using chloramphenicol andacetyl-CoA concentrations of 3.5 μM and 2.5 mM,respectively.

Reaction Conditions

0.5-2 mg/ml for 90 min

Applications

For transfection of the cells with a fixed amount of plasmid (usually 2 or 5 µg)

References:

[1]: Leventis R, Silvius J R. Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1990, 1023(1): 124-132.

[2]: Wilschut J, Duzgunes N, Fraley R, et al. Studies on the mechanism of membrane fusion: kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents[J]. Biochemistry, 1980, 19(26): 6011-6021.

[3]: Silvius J R, Leventis R, Brown P M, et al. Novel fluorescent phospholipids for assays of lipid mixing between membranes[J]. Biochemistry, 1987, 26(14): 4279-4287.

[4]: Gorman C M, Moffat L F, Howard B H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells[J]. Molecular and cellular biology, 1982, 2(9): 1044-1051.