Cell transfection is a fundamental molecular biology technique involving the artificial introduction of exogenous nucleic acids, such as DNA or RNA, into eukaryotic cells. This process bypasses the cell’s natural defense mechanisms to deliver genetic material, allowing it to be expressed. Transfection objectives range from studying gene function and regulation to producing recombinant proteins for research or therapeutic applications. Success requires selecting the appropriate delivery method and confirming that the genetic material is functional within the cell.
Chemical-Based Transfection Methods
Chemical transfection relies on reagents that form protective complexes with the negatively charged nucleic acid, enabling passage across the cell membrane. The most widely used approach utilizes cationic lipids, known as lipofection. These synthetic lipids possess a positively charged head group and a hydrophobic tail, allowing them to spontaneously assemble into structures.
The interaction between the cationic lipids and nucleic acids forms a lipoplex, which neutralizes the overall charge and facilitates attachment to the cell surface. The lipoplex is taken into the cell primarily through endocytosis, where the cell membrane engulfs the complex. Once inside the endosome, the lipoplex must escape into the cytoplasm before degradation occurs.
A typical lipofection protocol begins by separately diluting the nucleic acid and the lipid reagent in a serum-free medium, such as Opti-MEM. This step is necessary because serum proteins can interfere with complex formation. The diluted components are then gently mixed and incubated at room temperature for 10 to 30 minutes to allow the lipoplexes to fully form.
Following incubation, the formed complexes are added directly to the cultured cells, which are maintained in complete growth medium. Another prominent chemical method employs cationic polymers, notably Polyethylenimine (PEI). PEI is favored for large-scale production due to its cost-effectiveness, condensing the nucleic acid into positively charged particles. Release from the endosome is facilitated by the “proton sponge effect.”
Physical Transfection Techniques
Physical transfection methods bypass chemical carriers by using mechanical or electrical force to temporarily disrupt the cell membrane, creating direct pathways for nucleic acid entry. Electroporation is a highly efficient and versatile physical technique applicable to a wide variety of cell types, including those resistant to chemical methods. This technique involves exposing the cells and nucleic acid to a precisely controlled high-voltage electrical pulse.
The electrical field generates a change in potential across the cell membrane, momentarily destabilizing the lipid bilayer. This destabilization leads to the formation of transient, nanoscale pores, a phenomenon known as electropermeabilization. Nucleic acid molecules in the surrounding solution enter the cell through these pores, driven partly by the electric field.
Success in electroporation depends on optimizing specific electrical parameters, including the field strength and the duration of the pulse. If the parameters are too aggressive, the pores may become permanent, leading to significant cell death; if too mild, low transfection efficiency results. The process requires specialized equipment, including an electroporator device and disposable cuvettes with integrated electrodes to deliver the pulse.
Once the pulse is delivered, cells are immediately transferred back to their normal growth medium to recover, allowing temporary pores to reseal. A different physical method is microinjection, which uses a fine glass needle to manually inject nucleic acid directly into the cytoplasm or nucleus of individual cells. While microinjection yields high efficiency, it is a low-throughput technique reserved for delicate cells or specific applications like generating transgenic organisms.
Post-Transfection Verification and Selection
Once nucleic acid is introduced into the cells, the next stage confirms successful delivery and expression, which is categorized as either transient or stable. Transient expression occurs when the nucleic acid remains in the cytoplasm or nucleus without integrating into the host genome. This leads to expression for a limited time, typically 24 to 96 hours.
Verification of transient expression often employs reporter genes, which are sequences encoded alongside the gene of interest to produce an easily detectable signal. Common reporters include Green Fluorescent Protein (GFP), allowing researchers to visualize transfected cells using a fluorescence microscope. Luciferase is another reporter, an enzyme that generates a measurable light signal upon adding a specific substrate, providing a quantitative readout of expression levels.
Stable expression requires the foreign DNA to integrate into the host cell’s chromosome, ensuring the genetic material is passed to daughter cells during division. To isolate these rare, stably modified cells, a selection process uses a selectable marker gene co-transfected with the gene of interest. This marker typically confers resistance to a cytotoxic agent, such as an antibiotic.
After recovery, cells are cultured in a medium containing the selective agent, such as Geneticin (G418), puromycin, or hygromycin B. Only cells that have successfully integrated the resistance gene will survive and proliferate; non-transfected or transiently expressing cells will die. The selective agent concentration must be determined for each cell line to ensure only stable transfectants are isolated, establishing a permanent, genetically modified cell line.