Viromer® Transfection Results

What is transfection?

The technique can be used to express exogenous nucleic acids (DNA or RNA) in other cells. Plasmid DNA (pDNA) transfection is often the method of choice, lending itself to an array of gene expression or editing studies, by inserting the DNA of interest into the host’s nucleus where it is transcribed. While DNA may be more popular, using RNA opens up exploration of protein down-regulation with RNA interference (RNAi), or blocking microRNA (miRNA) activity, without the worry of modifying the host’s genome. Transfection using mRNA tends to be more efficient than DNA, but the transfected cells tend to produce less protein due to the limited lifespan of mRNA. These different approaches can be used to generate stable, or transient transfections, depending on your needs.

Cells that have been transiently transfected express the exogenous gene without incorporating it into the genome. This means that the cells will not continue to express the gene of interest through subsequent replications. Transient transfection will persist for just a few days, after which the exogenous gene is lost through mitosis or degradation.

If long-term exogenous gene expression is required, stable transfection methods can be employed to generate a permanent cell line. Cells are first transfected transiently with the gene of interest, and then co-transfected with a marker gene. A marker gene that offers a selectable advantage, such as resistance to an antibiotic or toxin, is typically used. So after transient transfection the stable method works by adding the antibiotic/toxin to the cell culture. It becomes easy to identify only those that have integrated the marker gene and in all likelihood the gene of interest, into their genomes.

Viral transfection

This stable method is known as transduction and uses a virus carrier for the delivery of exogenous genes or probes. The viral vectors are selected based upon the specific application, but usually fall into one of four categories.

Adenoviruses: double-stranded DNA viruses primarily used for transient transfection and limited to 8 kb of insert in dividing and non-dividing cells.

Adeno-associated viruses: single-stranded DNA viruses that are used for stable transfection and integrate at a specific chromosomal site in dividing and non-dividing cells. These have a limited packaging capacity at just a 5 kb insert.

Retroviruses: viruses with RNA genomes capable of randomly inserting double-stranded DNA copies of their genome into the chromosomes of dividing cells. They are used in stable expression experiments and can carry genetic material up to 8 kb.

Lentiviruses: a type of retrovirus capable of being used in both dividing and non-dividing cells for stable transfection with up to 9 kb of insert.

Viral transfection has a very high gene delivery efficiency (95–100%), is a relatively simple procedure and can be used in both in vivo and in vitro settings.

Mechanical transfection

These methods deliver nucleic acids to a host cell by physically manipulating the cell. Electroporation for example, is a widely used and highly efficient transfection technique. It works by exposing the cells to an electric field in order to cause a temporary destabilization of the cell membrane in the form of tiny holes known as electropores. While the electric field is applied, nucleic acids can move through these pores. When the field is switched off, the pores close, leaving the exogenous nucleic acids inside the host cell. This is a simple transfection method that can be applied to a range of cell types, but is prone to high cell mortality unless correctly implemented.

Alternatively, microinjection can transfect the nucleic acids directly into the nucleus of the host cell. However, this process is both time-consuming as technically demanding.

Chemical transfection methods

Chemical transfection can be one of the most cost-effective options. These include the use of reagents such as DEAE-dextran, calcium phosphate, cationic polymers or lipids (lipofection). Chemical methods of transfection take advantage of the well-defined chemistry of nucleic acids to either generate a precipitate that host cells will take up or that can be encouraged to fuse with and cross the cell membrane. These techniques are usually inexpensive, efficient and versatile. Their limitations come in the form of sensitivity to parameters such as pH and possible inconsistencies between reagents.

Hybrid transfection methods

Finally, there are hybrid or particle-based methods such as magnetofection or biolistic transfection. Magnetofection, uses a magnetic force to drive nanoparticles that have been associated with the exogenous nucleic acids into the host cell. This is rapid and has a high efficiency, yet remains a relatively new technology that requires adherent or immobilized cells. Biolistic particle transfection is similar but instead of a magnetic force, it fires nucleic acid-coated micro-particles at a high velocity through the host cell membranes.

Regardless of the technique used, there are still a number of points to consider when attempting to transfect cells with either DNA or RNA. Purity, cell viability and protocol optimization all need to be controlled in order to maximize the transfection efficiency.