Transfection, Template and Routes of DNA Delivery
Transfection
One of the most frequently used cell biology techniques is the process of introducing foreign nucleic acids, such as DNA, or small RNA (e.g. miRNA or siRNA) into eukaryotic cells via transfection. Transfections are incorporated into experiments to better understand functional aspects of cells with the end goal of developing a therapeutic strategy that includes DNA vaccines, gene therapy, molecular medicine, and other types of drugs.
As the name indicates, DNA transfection is a method to insert exogenous DNA into host cells. With DNA transfection, researchers aim to study gene expression, gene regulation and protein synthesis.
Template
With all the variety in transfection methods and protocols, selecting the appropriate reagent is vital in achieving optimum transfection efficiency. It’s important to consider factors like the cell type and culture conditions in order to determine which transfection reagent will work the best. Cells that are harder to transfect (primary cells, stem cells, suspensions, etc.) will require specific reagents that are suitable for them. Different transfection methods can also be used depending on the experimental objectives.
Most often the DNA transfected into cells is plasmid DNA, which is a construct that incorporates a functional promoter, correct orientation of the inserted DNA, and an antibiotic resistance gene for clonal selection (these cloning services are also commercially available, see cloning services). For successful expression of the gene insert, the cloning construct must have a compatible promoter, must encode the gene of interest, as well as contain a reporter gene that allows the researcher to select cells that are expressing the gene (e.g. B-Gal, GFP, RFP).
The amount of DNA used in transfection experiments is dependent on the type of DNA transfected, along with the cell line that is being transfected. Typical protocols for adherent cells suggest testing 1 to 10 μg of purified, plasmid DNA, but the amount needed may vary based on the transfection system and efficiency. It is worth noting that increasing the amount of transfected DNA may not necessarily increase the efficiency of the transfection.
Routes of DNA Delivery
Virus Mediated
Viral transfection takes advantage of the natural behavior of viral viruses to insert their genetic material into a suitable host cell. Regarding transfection, the DNA sequence to be inserted is integrated into a part of a viral genome. The cells to be transfected are then exposed to the viruses containing plasmid DNA. Although viral transfection is sophisticated and shows remarkably high efficiency, the application in humans has tough hurdles to clear. Finding viruses that do not cause additional diseases in humans yet are potent enough to force their genome into human cells is a difficult task. Most viruses must be carefully monitored to maintain acceptable biosafety levels. There are also several limiting factors that have to be acknowledged, like host-cell specificity, cell-line packaging, and the lytic nature of viral vectors. Adeno-associated viruses (AAVs) are promising in this respect with the caveat that producing them in large quantities is a challenge. Considering viruses are notorious for causing diseases, there is a general discomfort about the idea of using them for medical treatment, and as such, non-viral modes of DNA delivery are also being developed.
Lipid Transfection (Cationic)
Cationic lipid transfection systems offer great benefits over other systems. Lipid reagents are preferred when large amounts of DNA are to be inserted. Cationic lipid transfection uses lipids consisting of molecules with one hydrophobic end and a positively charged hydrophilic end. Negatively charged DNA molecules exposed to cationic lipid systems are attracted to the positively charged hydrophilic ends, which form complexes known as lipoplexes. Protected within lipoplexes, DNA molecules can gain entry through the phospholipid bilayer of the cell membranes.
Cationic lipids combined with specific ligands may enable therapeutic DNA molecules to be delivered to only the diseased cells, leaving healthy organs unaffected. This desired feature has profound applications in devising tumor-targeting cancer treatments.
Injected DNA
Injecting pure DNA sequences into cells is one of the most straight-forward transfection techniques. Physical methods of transfection are usually used to achieve bulk transfection or single cell transfection. Microinjection can minimize costs by controlling the amount of injected material administered, while also being applicable to many different cell types. However, the incorporation rate of the transfected DNA is far lower than other procedures and direct injection requires advanced equipment. Gene guns can be used to blast heavy metal particles coated with plasmid DNA into the target cells. Though used primarily in plants, gene guns are a potential candidate for delivering DNA in animal cells (https://www.nature.com/articles/srep24127).