Available transfection technologies are broadly classified into three groups: chemical, physical, and biological. No one transfection method can be applied to all cell types and experiments
Therefore, the ideal transfection approach should be selected based on your cell type and experimental needs. Specifically, the ability to deliver molecules into cells varies by payload size and cell type. For example, primary and stem cells are typically harder to transfect than more common cell lines. In addition, the method of transfection used should have high efficiency, low toxicity, and minimal effects on normal cell physiology, all while being easy to use and reproducible .
Selecting the right method of transfection
Explore transfection methods and select the method that is best for your cells and experimental application.
Table 1. Important factors for selecting the right transfection method
|Selection criteria||Cationic lipid-based|
|Efficiency: easy-to-transfect cells||+++||+++||+++|
|Efficiency: hard-to-transfect cells||++||+++||+++|
|Delivery of large payload (>7 kb)||++||+++||++|
|Ease of use||+++||+++||+|
|Cost per reaction||+++||++||+|
+++ Excellent for most applications; ++ Good for some applications; + Least recommended, but may be appropriate for some applications.
Chemical gene delivery
Chemical gene delivery methods use carrier molecules that neutralize or impart a positive charge onto nucleic acids.
Cationic lipid transfection
Cationic lipid transfection is one of the most popular methods and can yield high transfection efficiencies in a wide variety of applications and cell types. In addition, this method is versatile, with reagents available for delivery of DNA, RNA, or protein. Specifically, these reagents spontaneously form nucleic acid-cationic lipid reagent complexes that are taken up by the cell via endocytosis.
Calcium phosphate precipitation
Calcium phosphate precipitation is an easily available and inexpensive transfection method that can be used in many cell types. In this method, calcium phosphate facilitates binding of DNA to the cell surface for uptake via endocytosis.
DEAE-dextran transfection, one of the earliest chemical transfection methods, is relatively simple to perform and low in cost. In this method, the DEAE-dextran molecule forms a positively charged complex with the nucleic acid that can bind the cell membrane and enter via endocytosis or osmotic shock.
Cationic polymer transfection
Cationic polymers, which may vary in their degrees of transfection efficiency, are completely water soluble and work by allowing the formation of nucleic acid-polymer complexes. These complexes can adhere to the cell membrane for uptake via endocytosis.
Figure 1. Mechanisms of chemical transfection. The positive surface charge of the reagent-DNA complex enables attachment to the cell surface and entry via endocytosis. Following escape from the endosome, DNA payloads are translocated to the nucleus for transcription whereas mRNA payloads can be translated directly in the cytoplasm.
Physical gene delivery
Physical gene delivery methods allow nucleic acids to be delivered directly into the cytoplasm or nucleus of the cell without the use of chemical carrier molecules.
Electroporation is a popular physical method of transfection that uses an electrical pulse to create temporary pores in cell membranes through which nucleic acids can pass. This method can be used for the rapid transfection of a large number of cells and is applicable in a range of settings, including clinical studies.
Other physical delivery methods
Other physical gene delivery methods differ from electroporation but still facilitate the direct transfer of nucleic acids into cells without using carrier molecules. These include biolistic particle delivery, in which nucleic acid-coated particles are projected into cells, microinjection, in which a needle is used to directly inject nucleic acids into cells, and laser-mediated transfection, where a laser pulse creates cellular pores.
Figure 2. Mechanism of electroporation. When an electric field is induced across the cell membrane, multiple pores form that allow entry of the payload into the cell. Following electroporation, the cell membrane recovers, and the payload is distributed in the cytoplasm and nucleus.
Biological gene delivery
Biological transfection methods utilize genetically engineered viruses to transfer nucleic acids into cells.
In viral-mediated transfection, also referred to as transduction, viruses serve as vectors, carrying genes into eukaryotic cells. This method is often used in hard-to-transfect cell types not amenable to other transfection methods and is commonly used in clinical research.
Figure 3. Mechanism of viral transfection. (1) Packaging cells are transfected with three to four plasmids encoding the gene of interest and viral proteins. (2) The virus is assembled in the packaging cell, harvested, and purified. (3) The virus is used to transduce target cells, releasing the gene of interest. (4) In this example, RNA from the lentiviral vector is reverse-transcribed to DNA, which integrates into the host genome for recombinant protein expression.
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