While various approaches can be used to stably or transiently transfect genetic material, they broadly fall into three categories – physical, viral, and chemical methods. Of the three main types of transfection, chemical-mediated transfection is the most widely used in contemporary research for its ease and cost-effectiveness.
Physical transfection methods, such as electroporation, microinjection, and biolistic particle delivery, rely on a diverse set of physical tools that use mechanical or electrical forces to deliver exogenous nucleic acids. Microinjection utilizes a micromanipulator and microscope to directly insert nucleic acids into the cytoplasm or nucleus. Although time-consuming and technically challenging, microinjection affords users the ability to control the amount and timing of injected materials delivered and is suitable for various cell types. Biolistic particle delivery, also called particle bombardment, uses a ballistic device to shoot nucleic acids coated with gold particles into cells at a high velocity. This approach may not be as precise as microinjection, but it offers a simple method to both transient and stable transfection, can be used on various tissue and cell types, including plants, and can co-deliver multiple plasmids with high frequencies of cotransfromation.
Of the various types of physical transfection, electroporation is the most widely used. In this method, an electrical field is applied to cells, which perturbs the cell membrane resulting in the formation of transient pores that facilitate the entry of exogenous material. Electroporation is applicable for transient and stable transfection of all cell types, and under optimal conditions, it can be adapted for high-throughput transfection. Although physical transfection is advantageous for its low cytotoxicity and ability to deliver genetic material rapidly and directly, these methods are relatively harsh on cells causing disruption to the cellular membrane and often results in substantial cell death.
Viral-mediated transfection, also known as transduction, is widely used in clinical research for its high in vivo transfection efficiency and sustained gene expression without severely affecting cell viability. In contrast to chemical transfection methods, no transfection reagent is required. Instead, the viral vector infects the cells and delivers the genetic material directly to the nucleus. Common viral delivery systems include adenoviral, oncoretroviral, lentiviral, baculovirus, and vaccinia virus-based vectors. Even though viral-mediated transfection is highly effective, the major concerns associated with its usage are immunogenicity, cytotoxicity, and low packaging capacity.
Chemical-mediated transfection relies on electrostatic interactions to deliver exogenous nucleic acids into cells. In this method, electrostatic forces between the cationic charges on the lipid or polymer groups of transfection reagents and the negatively charged phosphates of nucleic acids cause the two substances to associate, resulting in positively charged transfection complexes. These cationic transfection complexes, in turn, interact with negatively-charged glycoproteins, such as heparan sulfate proteoglycans, expressed on the cell surface triggering the cellular uptake of exogenous nucleic acid via endocytosis or phagocytosis. Commonly used methods for chemical transfection include calcium phosphate, cationic polymer, and cationic lipid transfection (see Table 1).
Table 1.Overview of Chemical transfection methods