Chemical- and Viral-Based Transfection Methods

• Cationic lipids are amphiphilic molecules that have a positively charged polar head group linked, via an anchor, to a nonpolar hydrophobic domain generally comprised of two alkyl chains
• Structural variations in the hydrophobic domain of cationic lipids include the length and the degree of non-saturation of the alkyl chains
• Electrostatic interactions between the positive charges of the cationic lipid head groups and the negatively charged phosphates of the DNA backbone are the main forces that allow DNA to spontaneously associate with cationic lipids

The protocol involves mixing DNA with calcium chloride, adding the mixture in a controlled manner to a buffered saline/phosphate solution, and allowing the mixture to incubate at room temperature.

This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken up by the cells via endocytosis or phagocytosis.

Intercalation of Ca⁺⁺ ions.

Protocol

Solution A: DNA in calcium solution
Solution B: 2x Hanks buffered saline solution

• Add solution A to solution B while vortexing
• Incubate 20–30 min. Apply the solution to the subconfluent cell culture
• Incubate 2–12 hr. Replace the solution with complete growth medium
• Assay for transient gene expression or begin selection for stable transformation

Cationic polymers differ from cationic lipids in that they do not contain a hydrophobic moiety and are completely soluble in water. Given their polymeric nature, cationic polymers can be synthesized in different lengths, with different geometry (linear versus branched). The most striking difference between cationic lipids and cationic polymers is the ability of the cationic polymers to more efficiently condense DNA.

There are three general types of cationic polymers used in tranfections:

• Linear (histone, spermine, and polylysine)
• Branched
• Spherical

Cationic polymers include polyethyleneimine (PEI) and dendrimers.

DEAE-dextran is a cationic polymer that tightly associates with negatively charged nucleic acids. The positively charged DNA:polymer complex comes into close association with the negatively charged cell membrane. DNA:polymer complex uptake into the cell is presumed to occur via endocytosis.

Protocol

Solution A: DNA (~1–5 µg/ml) diluted into 2 ml of growth medium with serum containing chloroquine
Solution B: DEAE-dextran solution (~50–500 ug/ml)
Solution C: ~5 ml of DMSO
Solution D: Complete growth medium

• Add solution A to solution B, then mix gently
• Aspirate cell medium and apply the mixed A and B solutions to the subconfluent cell culture. Incubate the DNA mixture for ~4 hr; check periodically for cell health
• Aspirate the supernatant
• Add solution C to induce DNA uptake
• Remove DMSO and replace with solution D; assay for transient gene expression

Positively charged amino groups (termini) on the surface of the dendrimer molecule interact with the negatively charged phosphate groups of the DNA molecule to form a DNA-dendrimer complex.

The DNA-dendrimer complex has an overall positive net charge and can bind to negatively charged surface molecules on the membrane of eukaryotic cells. Complexes bound to the cell surface are taken into the cell by nonspecific endocytosis. Once inside the cell, the complexes are transported to the endosomes.

• DNA is protected from degradation by endosomal nucleases by being highly condensed within the DNA-dendrimer complex.
• Amino groups on the dendrimers that are unprotonated at neutral pH can become protonated in the acidic environment of the endosome. This leads to buffering of the endosome, which inhibits pH-dependent endosomal nucleases.

Dendrimer molecule.

Magnet-mediated transfection uses magnetic force to deliver nucleic acids into target cells. Therefore, nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid-particle complexes towards and into the target cells, where the cargo is released.

Magnet-mediated transfection.