Transfection is a molecular biology technique where foreign DNA is introduced into higher eukaryotic cells. The DNA typically encodes a gene that expresses a protein or RNA product when placed in the cell. The DNA is typically placed into a plasmid that can express the protein product in a certain cell line. Levels of expression can vary from high to low depending on the type of plasmid used. Plasmids can either express their products constantly or can be induced to express, as seen with a tet-regulated system. The idea for transfection was originally discovered in the 1960s. After the technology for cloning DNA was discovered, it was easy for any sequence of DNA to be introduced into cells via transfection. Transfection is analogous to a technique known as transformation, where foreign DNA is placed into bacteria or yeast cells.
There are many uses for transfection, such as:
- Creating large amounts of a particular protein from the gene that encodes it. The protein can then be isolated and purified for other techniques.
- Introducing a protein into a cell that may lack it to get a better idea of that protein's function.
- Conversely, inhibiting the expression of a protein or mRNA already present in the cell to better understand its function (see antisense peptides and RNAi).
- Analyzing how a gene is regulated in the cell (see reporter assay)
Cells are often cotransfected, where two different plasmids are introduced into the cell simultaneously. This technique is used to monitor the efficiency, or what percentage of cells take up and express the DNA, of a transfection. The first plasmid contains the DNA of interest while the second plasmid encodes a gene like green fluorescent protein, or GFP, that produces a fluorescent protein. Cells that have been cotransfected can then be analyzed under a microscope to see what percentage are fluorescent. The percentage of cells that are fluorescent generally corresponds to the percentage of cells that were transfected with the DNA of interest.
Transfections can be divided into two kinds, transient and stable:
- Transient: The plasmid is temporarily inserted into the cells. It is not incorporated into the genome of the cell, but instead is present as a separate episome. The plasmid is eventually lost as the cell grows and divides. Protein expression levels are often initially higher than stable transfections because there is a higher plasmid to cell ratio. However, over time the expression decreases as the plasmids are lost from the cells. Transient transfections are quick and easy. Results are fast since the cells are harvested between 1 to 4 days after transfection. This method is especially useful if prolonged expression of a certain protein is toxic to cells.
- Stable: The plasmid is permanently integrated into the cell by inserting itself into the cell's genome. This means the DNA will remain in the cell even as it divides. This integration is a rare event and the cells that contain the plasmid DNA in the genome must be selected for by using a marker found on the plasmid. This marker provides the cell resistance to certain antibiotics. Common markers include the neomycin resistance gene (neo), the thymidine kinase gene (TK), and the hygromycin B phosphotransferase gene which makes the cells resistant to the antibiotics G418, aminopterin, and hygromycin B, respectively. The cells are treated with the antibiotic and only those that have been stable transfected will survive. Stable cell lines have an overall protein expression level that is much lower than with transient transfections. This method will provide stable, long term expression, but it can be difficult to obtain a stable line and screening often takes several months.
Since cells will not simply absorb the DNA, there are several different techniques used to insert the DNA into cells. Commonly used techniques, listed in order of their discovery, include:
- Calcium Phosphate: This method was discovered by Graham and Van der Eb in 1972. CaCl2 is mixed with a solution containing the DNA. This creates calcium phosphate (CaPi), which is insoluble and precipitates. The DNA is trapped within the calcium phosphate and precipitates with it. This DNA precipitate is mixed with cells where it binds to the cell membrane and is absorbed, presumably via endocytosis. This technique is cheap, easy, and can be used for both transient and stable transfections, but has a low efficiency.
- DEAE-dextran: This technique was discovered by Vaheri and Pagano. Since DNA is negatively charged it cannot get through the positively charged cell membrane. DEAE-dextran is a positively charged polymer that binds strongly to the negatively charged phosphate groups on the DNA. The overall complex is positively charged and it can bind to the cell membrane, where the DNA is absorbed. This method is simpler and easier than the calcium phosphate method, but can only be used for transient transfections.
- Liposome-mediated: Artificial liposomes were used in the 1980s to transfer DNA into cells that were unresponsive to the above two methods. Felger and colleagues developed synthetic positively charged lipids that made this process easier. The concept of this technique is very similar to the DEAE-dextran method. The DNA is surrounded by a positively charged lipid sphere that masks the negative charge. This sphere fuses to the cell membrane and the DNA is absorbed. This method is simple, fast, and efficient for both transient and stable transfections, however the reagents are expensive.
- Electroporation: This method was first developed in 1982 in order to be able to transfect a larger variety of cell lines. The cells are mixed with the DNA and a high voltage of electricity is applied. This is thought to form holes in the cell membrane, allowing the DNA to enter. This method is very fast, simple and efficient for both transient and stable transfections. However, this method is very abusive to cells, as generally 50-70% of them die from the shock. Care must be taken to use the right voltage for maximum efficiency with minimal cell death. Electroporation can only be used on suspension cells (those that float in the medium), not adherent cells (those that stick to the bottom of a flask).
Less common techniques for introducing DNA:
- Viral vectors: Viruses such as adenovirus or retrovirus are used to introduce the DNA. A small part of the virus's genome is replaced by the DNA of interest. The virus then infects the cells and delivers the DNA. Creating a vector that contains the DNA of interest is very time consuming, but the process is extremely efficient.
- Microinjection: The DNA is injected into each cell using a microscopic needle. This technique is commonly used for cloning and the creation of knockout organisms. Microinjection is a very labor-intensive technique but very efficient. This method is not useful for transfecting a large amount of cells.
- Gene guns: The plasmid DNA is attached to tiny gold beads and shot into the cells using a gene gun. Both the gun and its ammunition are very expensive, making this technique only practical in mammalian cells if every other technique has failed. However, this method is commonly used in plant cells to get the DNA through the tough cell wall.
The efficiency of these techniques varies widely. Efficiency depends on many factors, including the transfection technique, type and condition of the cells, and purify of the plasmid. All types of transfection are more efficient when the cells are actively dividing. This is because the nuclear membrane is absent during cell division, making it easier for the foreign DNA to access the host's genome.
For more reading:
- Current Protocols in Molecular Biology, Volume 2, Section 9, 1998