Cell Transfection Wiki: Transfection protocols, stable transfection, new generation of transfection reagents
Cell transfection occurs when nucleic acids (or sometimes other molecules such as proteins) are deliberately introduced into cells, often using a non-viral method in eukaryotic cells. Sometimes “transfection” is used to describe other processes, such as “transformation” where non-viral DNA is inserted into bacteria and non-animal eukaryotic cells, or “transduction” where viruses are used to transfer DNA into cells. The process of transfection typically requires opening pores in the cell membrane to allow the molecules being transfected to cross into the cell. This can be done in a number of ways: chemically, using electricity, or by putting the molecules in liposomes. Typically the desired gene is coupled with a reporter gene, such as one that confers resistance to an antibiotic, to allow the elimination of cells that do not contain the desired gene post-transfection.
Chemical transfection can be accomplished in a number of ways. One of the most inexpensive techniques utilizes calcium phosphate precipitation. A calcium chloride solution containing the DNA intended for transfection is mixed with HEPES -saline buffer with phosphate ions, causing precipitation of the insoluble calcium phosphate, in the process binding the DNA on the precipitate surface. The precipitate (in suspension) is then mixed with the cells intended for transfection, and the cells take up the precipitate, including the DNA. Another method involves the use of dendrimers, highly branched organic compounds that bind the DNA and bring it into the cell. Cationic polymers can also be used for chemical transfection, as they bind the negatively-charged DNA and then the entire polymer is endocytosed into the cell.
Electroporation is another common technique for transfection. It consists of the external application of an electrical field, which increases the permeability of the cell membrane. Electroporation is more effective than chemical transfection, but it does require careful handling of the cells until the inserted DNA is established in the cell line.
Liposomes are small membrane-bound capsules made of polymers similar to those that make up the cell membrane. When constructed to contain DNA, they can fuse with the cell membrane and release that DNA into the cells. This is often called “lipofection”.
In addition, there are a number of less common transfection methods, including:
- Cell squeezing: invented by Robert Langer’s group at MIT, this involves a gentle squeezing of the cell using a microfluidic device to induce transfection.
- Sonoporation: similar to electroporation, but utilizing high-intensity sound instead of electric energy.
- Optical transfection: using a high-focus laser to generate a temporary opening in the cell membrane of a single cell.
- Protoplast fusion: treats cells with lysozyme to remove cell walls, and then uses a secondary technique of fusion (polymers or electroporation, for example) to fuse the protoplast containing the DNA into the cell.
- Impalefaction: using a nanofiber with DNA bound to its surface to insert the DNA into the cell.
- Gene gun or particle bombardment: DNA is coupled to a nanoparticle of a biocompatible solid and then “shot” into the nucleus of the target cell.
- Magnetofaction: this method uses magnetic nanoparticles associated with DNA, which are then driven into the target cells using a magnetic field.
There are two ways that transfection can proceed in the cell, either in a transient manner or as a stable transfection.
- DNA is introduced into the cell nucleus, but does not get integrated into the chromosomes
- High levels of target protein expression, due to large number of gene copies present
- Transfected gene is expressed for a finite amount of time, typically several days, and is then removed through cell division or other means
- mRNA, siRNA and miRNA can also be used for transient transfection, and do not need to be inserted into the nucleus to be effective
- Generally uses chemical or electroporation methods
- Starts similarly to transient transfection, however a small number of cells integrate the inserted DNA into the chromosome or maintain it as an episome
- Stable transfection is a rare event, and cannot always be predicted
- Generally more successful with linear DNA rather than supercoiled DNA
- miRNA and siRNA can only undergo stable transfection when they are delivered as shRNA using a DNA vector; no RNA molecules can be stably transfected on their own
- Typically uses viral or microinjection methods
- Has a risk of non-specific integration into chromosomes
Stable transfection is a much more complicated process than transient transfection. However, when long-term gene expression is necessary, in cases like gene therapy, large scale protein production, long-term pharmacological experiments, or studies of genetic regulation on long timescales, the extensive effort is undertaken to produce stably transfected cell lines.
Traditionally large amounts of recombinant proteins are produced with stably transfected mammalian cells to encourage proper folding and the correct post-translational modifications. However, recent improvements in transient transfection and cell culture methods (particularly growth in suspension) have increased the ability of researchers to use transiently-transfected cells for this purpose. In particular, suspension growth of HEK293 and CHO cells has been used along with transient expression to create large quantities of usable recombinant proteins expediently.
Some examples of scientific studies that utilize cell transfection include:
- Manufacturing high-titer helper-free retroviruses: This study utilized transient transfection in 293T cells to express retroviral packaging functions. Using transient transfection avoiding the time consuming process of finding a single stably-expressing clone, and produces a high titer of retrovirus within 72 hours.
- Transfection of protein-producing DNA using lipid transfection reagent: Researchers used a cationic lipid transfection reagent to study how transient transfection performed in HEK293 and CHO cells. Both types of cells were able to produce acceptable titers of GFP and secreted IgG antibodies when transfected with the corresponding gene. In addition, they evaluated production of erythropoietin and factor IX in the same cell types.
- Transfection via laser: Cells in this study were successfully transfected at a high rate using femtosecond high intensity near-IR laser pulses to perforate the cell membrane. This technique allows for high efficiency as well as high rates of cell survival and leaves the cell largely intact.
- Cationic polymer transfection: Researchers in this study used a cationic polymer to transfect HEK293 cells grown in suspension. The cationic polymer (both linear and branched) was highly successful at transfection, and is a scalable procedure. The desired proteins were produced at significant levels and the cells did not have some of the typical undesired properties such as medium-conditioning effects previously seen in the use of polymeric transfection reagents.
- There commercially available transfection services provided by GLP certified research laboratories