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Overview of Transfection Methods |
Transfection methods are broadly categorized into three main types: chemical, physical, and biological. No single transfection method or technique can be applied to all cell types and experiments.
Therefore, the ideal transfection technique should be selected based on your cell type and experimental needs. Specifically, the ability to transfect cells with nucleic acids 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 chosen method of transfection should be low in toxicity, minimally disruptive to normal cell physiology, and have high efficiency. Ease of use and reproducibility are also important when determining which transfection technique you will use [1].
How to select the right method of transfection
To choose the right transfection method, consider your cell type, payload, and experimental goals. See the table below to guide your selection by efficiency, viability, cost and more.
Table 1. Important factors for selecting the right transfection method
| Selection criteria | Cationic lipid-based Chemical method | Electroporation Physical method | Viral delivery Biological method |
|---|---|---|---|
| Efficiency: easy-to-transfect cells | +++ | +++ | +++ |
| Efficiency: hard-to-transfect cells | ++ | +++ | +++ |
| Cell viability | +++ | ++ | +++ |
| Delivery of large payload (>7 kb) | ++ | +++ | ++ |
| High-throughput suitability | +++ | ++ | +++ |
| Ease of use | +++ | +++ | + |
| Biosafety | +++ | +++ | + |
| Cost per reaction | +++ | ++ | + |
+++ Excellent for most applications; ++ Good for some applications; + Least recommended, but may be appropriate for some applications.
Chemical transfection
Chemical transfection (chemical gene delivery) methods use carrier molecules—typically cationic lipids or polymers—that form complexes with negatively charged nucleic acids, facilitating their uptake into cells.
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
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.
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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 transfection
Physical methods of transfection (physical gene delivery) allows nucleic acids to be delivered directly into the cytoplasm or nucleus of the cell without the use of chemical carrier molecules.
Electroporation
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
In addition to electroporation, there are other physical transfection methods including: microinjection, biolistic particle delivery, and laser-mediated transfection. Other physical gene delivery methods differ from electroporation but still facilitate the direct transfer of nucleic acids into cells without using carrier molecules. Microinjection achieves this by using a needle to directly inject nucleic acid into cells. In biolistic particle delivery, nucleic acid coated particles are projected into cells. Finally, laser-mediated transfection creates cellular pores with a laser pulse.
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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 transfection
Biological transfection methods utilize genetically engineered viruses to transfer nucleic acids into cells.
Viral transfection
Viral transfection, or transduction, uses modified viruses like lentivirus or AAV as vectors to deliver genetic material into eukaryotic host cells. Viral transfection (transduction) is often used in hard-to-transfect cell types not amenable to other transfection methods and is commonly used in clinical research.
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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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References
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