There are different transfection methods existing, all having their advantages and disadvantages. Below, we shortly describe the different transfection methods ranging from viral transfection, to chemical transfection methods like calcium-phosphate precipitation, lipofection and DEAE-dextran, as well as electroporation. We end this paragraph with our improved electroporation technology, which is also called Nucleofection.
Viral transfection (also called viral transduction) takes advantage of the natural ability of certain viruses to carry foreign genes into host cells. Depending on the virus used (adenovirus etc.), you can achieve either stable or transient transfection. Using viral methods for primary cell transfection and cell line transfection generally yields high efficiencies. But there are several challenges to overcome when using viral transfection. For instance, it is often a time-consuming process that raises biosafety considerations (depending on the type of virus being used). In addition, the size of your DNA or RNA insert is limited to about 10 kb for most viral vectors. This is small when compared to non-viral vectors, which can carry up to 100 kb. A range of viral vectors can be used for transfection, each of which has different advantages, disadvantages and applications.
If you’d like to learn more about the different viruses used, download our free whitepaper: ‘An Introduction to Transfection Methods – Technical Reference Guide’.
Calcium Phosphate Transfection
Transfection requires the delivery of material through the cell membrane, which is negatively charged. As nucleic acids such as DNA and RNA are also negatively charged, they repel each other, inhibiting their uptake by the cell. One way to overcome this challenge is to use positively-charged carrier molecules to ferry negatively charged substrates close enough to the cell membrane to be internalized via endocytosis. Calcium phosphate Transfection is one of the chemical transfection methods using this principle.
This technique involves mixing your DNA construct with calcium chloride in a buffered saline/phosphate solution. When the mixture is incubated at room temperature, DNA-calcium phosphate coprecipitates form. As these coprecipitates can adhere to the plasma membrane, they are thought facilitate endocytosis. Calcium phosphate transfection is widely used for both transient and stable transfection applications across a range of cell types, especially as the required reagents are affordable and accessible for most labs.
Unfortunately, this method can be toxic to cells (especially primary cells) and the transfection efficiency is relatively poor compared to most other methods. In addition, the reaction used to generate the coprecipitates is sensitive to slight changes in pH, temperature and buffer-salt concentrations, leading to unreliable results.
It is also possible to use diethylaminoethyl-dextran (DEAE-dextran) to facilitate transfection. DEAE-dextran is a polycationic derivative of dextran (a carbohydrate polymer). When mixed with the DNA, the resulting complex is carried into contact with the negatively-charged plasma membrane by the excessive positive charge provided by the polymer. As for calcium phosphate, this close proximity to the membrane is thought to facilitate endocytosis into the cell. While using DEAE-dextran is relatively simple and low cost, the transfection efficiency of the method is generally low for many cell types. It can also be cytotoxic and is not usually suitable for generating stable cell lines.
Lipofection is the most commonly-used chemical method of transfection. The approach involves combining cationic lipids with other molecules to create unilamellar liposome vesicles that carry a positive charge. The exact molecular mixture has varied over time, as lipofection methods have improved.
Regardless of the makeup of the vesicles, lipofection is designed to package negatively charge molecules like nucleic acids in a positively-charged vesicle, so that they can get closer to the cell membrane (where they are presumably taken up by endocytosis). As such, you must mix your construct or molecule of interest with the vesicle mixtures ahead of transfecting your cells.
The approach can be successfully used to transfect a wide range of cell types at relatively low cost (although cost and reaction conditions can increase as other polymers and antibody-conjugates are added). Furthermore, lipofection can transfect cells using DNA of all sizes to produce both stable and transient transfection, as well as deliver RNA and proteins into cells. The main drawback of the technique is the low transfection efficiency achieved when it is used for primary cell transfection, stem cell transfection and when working with suspension cell lines (mainly due to the fact that it can be cytotoxic and relies on cell division for success).
If you’d like to learn more about Lipofection and other chemical-based transfection methods, download our ‘Transfection Methods – Technical Reference Guide’ here.
Electroporation is a physical method of transfection that involves first suspending your cells and DNA construct in an electroporation buffer. High-voltage pulses of electricity are then applied to the mixture, which creates a potential difference across the cell membrane. This introduces temporary pores that allow the exogenous DNA to enter the cell. To successfully perform this technique, you usually need to test and optimize the duration and strength of the pulses against your specific cell type and construct.
As for all transfection methods, electroporation has its advantages and disadvantages. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. Furthermore, relatively high substrate amounts are required to achieve efficient transfection.