Transfection (one of the most recurrently used cell biology laboratory techniques) is introducing foreign nucleic acids, DNA and RNA, in eukaryotic cells. The objective of performing transfection is to better understand functionality and structure of cells. The ultimate goal of that research is developing therapeutic strategies for DNA vaccines, gene therapy, molecular medicine and drugs.
DNA transfection refers to the inserting exogenous DNA into host cells. Through a DNA transfection researchers aim to study gene expression and protein synthesis. Several DNA transfection methods are standardized since the development of this technology. DNA transfection methods include viral vectors, injectable naked DNA and nanoparticles. Liposome forming cationic lipids, polyethylenimines, chitosan, and polymers are also being tested for plasmid DNA transfections.
Viral transfection method takes advantage of a natural behavior of viruses to insert genetic material in proper host cell. For the transfection purposes, the DNA is partially made of a viral genome. Cells that need to be transfected are exposed to viruses with plasmid DNA. Despite the fact that viral transfection of DNA was demonstrated to be successful, its application in humans is replete with difficulties. Finding viruses that don’t cause diseases in humans and effective enough to force their genome into human cells is difficult. Adeno-associated viruses (AAVs) is promising in this respect, but producing it in large quantities is a challenge. There is a general discomfort with the idea of using viruses for medical treatment because viruses naturally cause deceases.
Due to the concern related to viral transfection, there are ongoing efforts to develop non-viral modes of DNA delivery. Technological advancements allowed the development of highly efficient DNA transfection molecules. Using cationic lipid transfection and injecting naked DNA systems are some of the instances of non-viral DNA transfection.
Cationic lipid transfections use lipid systems made of molecules with a hydrophobic end and a positively-charged hydrophilic end. Naturally, negatively charged DNA molecules (when exposed to cationic lipid systems) get attracted to the positively-charged hydrophilic ends and form composites known as lipoplexes. Protected with lipoplexes, DNA molecules safely enter through membranes’ phospholipid bilayer to be transfected. Cationic lipid systes offers significant benefits. They are preferred when massive amounts of DNA are inserted; they also cause low cell toxicity. When combined with specific ligands, catonic lipids enable therapeutic DNA molecules to enter the diseased cells leaving healthy organs unaffected. This feature has application in devising tumor targeting cancer treatment.
Physical transfection methods includes electroporation, microinjection and biolistic particle delivery. Electroporation is dependent on DNA concentration and type of cells used. Microinjection is commonly used to introduce DNA into single cells such as embryonic stem cells. However, the success rate of this method is lower than success rate of other DNA transfection procedures. Using micromanipulator and microscope, the DNA can be directly inserted into the cytoplasm or nucleus of individual cell. This is very time consuming, but results in high transfection efficiency. Biolistic particle delivery relies on microparticles carrying nucleic acid to introduce plasmid DNA into cells. Gene guns are used for blasting heavy metal particles covered with plasmid DNA to the target cells. Gene guns are a potential candidate for delivering DNA vaccines to the animals.
Compared to pioneers in the field of cell Biology, modern researchers have protocols, transfection reagents, kits and other products available. Scientists can focus on the areas of interest as they have general purpose and cell-line specific reagents (optimized to produce high efficiency transfection). Also, custom stable cell line generation contract research are commercially available (read more about RNAi custom services).
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