Transfection refers to the introduction of foreign DNA into a eukaryotic cell, and can be achieved by simple uptake of a precipitate mixture of calcium phosphate and the DNA to be transfected. Although this represents the least expensive of methods, it is usually not very effective; faster and more reliable DNA uptake is usually accomplished using specially-developed transfection reagents, which have been classified as either cationic, liposomal, or involving use of nanoparticles.
JetPEI™, a transfection reagent from Polyplus, is classified as a cationic polymer, with a positive charge on approximately 1 in 3 nitrogen species on its molecule. It is a linear water-soluble polymer, and is attracted to negatively charged phosphate groups on DNA with which it forms stable aggregates. The Nitrogen/Phosphate (N/P) ratio is a measure of the ionic charge on the jetPEI™/DNA complexes and is projected to be around 3. The higher the N/P ratio, the stronger the attraction to negatively charged proteoglycans at the cell surface, and the better the transfection efficiencies obtained. An N/P ratio of 5 is recommended by the company as the ideal one to use. At the cell surface the jetPEI™/DNA complexes enters the cell by endocytosis, where the jetPEI™ behaves as a proton sponge in the endosome, buffering the pH. This mechanism leads to endosome swelling and rupture, permitting the release of the jetPEI™/DNA complexes into the cytoplasm.
JetPEI™ is supplied by the company as a 7.5 mM solution, and is accompanied by a 150 mM NaCl sterile solution for dilution of the reagent and of the plasmid DNA to be transfected. A formula is provided in the manual to calculate amounts of the reagent to use for a desired N/P ratio, and amounts of DNA to be transfected. The actual procedure can be completed in 1 hour depending on the number of flasks/plates to be transfected, since incubation of the jetPEI™/DNA complex need only be for 15 -30 minutes at room temperature. However, the jetPEI™/DNA complex can be left in incubation for up to 2 hours, and it is claimed that this does not affect transfection efficiency, allowing plenty of time to dispense the complexes into the plates.
The major advantage of using jetPEI™ is that since it is not affected by the presence of serum, or antibiotics, it can be added directly to serum-containing medium. In our experience, we have found that at higher N/P ratios, we can obtain higher transfection efficiencies, up to 70% using enhanced green fluorescence protein (EGFP) as the transfection marker. However, marked cytotoxicity is also observed, and this is avoided by using a fraction of the recommended DNA and jetPEI™ amounts, while maintaining a high N/P ratio of ~ 8. As is also recommended in the protocol, aspirating the medium after a 2-4 hour incubation period after transfection also reduces toxicity. In our lab we use this product about 2-3 times/ week to transfect 96-well, 24-well, 12-well, as well as 6-well plates of ST14A cells and HEK-293 cells, but it is claimed by the company that the reagent can be used for more than a hundred established cell lines, as well as for primary ones.