Transfection is the process of introducing nucleic acids into mammalian or insect cells. Unlike bacterial transformation, which refers to bacterial uptake of DNA, transfection specifically describes delivery into eukaryotic cells.
Lipid-Mediated Transfection
Cationic lipid reagents (Lipofectamine, FuGENE, jetPEI) form liposomes that encapsulate negatively charged DNA. The positively charged liposomes fuse with the negatively charged cell membrane, releasing the DNA into the cytoplasm.
The lipid-to-DNA ratio is critical. Most reagents require optimization in a 2–4 µL lipid per µg DNA range. Transfection efficiency depends on cell type — HEK 293 and HeLa are highly transfectable; primary cells and suspension cells are more difficult. Serum in the medium can inhibit some lipid reagents. Most protocols recommend transfecting in reduced-serum medium (Opti-MEM) and replacing with complete medium 4–6 hours later.
Calcium Phosphate Co-Precipitation
DNA is mixed with CaCl₂ and added dropwise to a phosphate-buffered solution, forming a fine precipitate of calcium phosphate–DNA complexes. The precipitate settles onto the cells and is internalized by endocytosis.
This method is very inexpensive but sensitive to pH. The optimal pH (typically 6.95–7.05) must be determined empirically for each cell type. The precipitate must be fine and even — large aggregates reduce efficiency and increase toxicity. Calcium phosphate works well for adherent cell lines but poorly for primary and suspension cells.
Electroporation
A brief high-voltage pulse (100–300 V, 1–25 ms) creates transient pores in the cell membrane through which DNA enters. Electroporation works for nearly any cell type, including primary cells, stem cells, and suspension cells that resist chemical methods.
Commercial electroporation systems (Neon, Nucleofector, Gene Pulser) use optimized pulse parameters and proprietary solutions to achieve 70–90% efficiency in hard-to-transfect cells. The trade-off is significant cell death (20–50%), and careful optimization of cell density, DNA amount, and pulse parameters is required.
Viral Transduction
Recombinant viruses deliver genetic material with high efficiency to a broad range of cell types. The most common vector systems:
- Lentivirus: infects dividing and non-dividing cells and integrates into the genome, enabling stable long-term expression. The packaging capacity is ~8 kb.
- Retrovirus: infects only dividing cells. Used for stable expression in rapidly proliferating cells.
- Adeno-associated virus (AAV): non-integrating, low immunogenicity, good for in vivo delivery. Packaging capacity is ~4.5 kb.
- Adenovirus: high-titer, infects many cell types, but triggers an immune response. Used for transient, high-level expression.
Stable vs. Transient Transfection
Transient transfection: the DNA remains episomal and is diluted by cell division. Expression peaks at 24–48 hours and lasts 2–7 days. Used for short-term experiments, reporter assays, and protein production.
Stable transfection: a selectable marker (puromycin, G418/hygromycin resistance) is co-transfected, and cells that integrate the DNA into their genome are selected over 7–14 days. The resulting stable cell line maintains expression indefinitely.
