Transfection is one of the most common techniques in biological research. This term refers to the introduction of genetic materials such as DNA and RNA into mammalian cells. The goal of transfection is usually to investigate the effects of expression/silencing of target genes. Current transfection techniques can be broadly divided into viral, chemical, and physical methods.
As the name suggests, this method utilizes viruses to deliver genetic materials into cells. The most common viruses used are adeno-associated virus and lentivirus. There are also other viral vectors, such as retrovirus HIV and herpes simplex viruses.
- 95-100% success rate.
- Viral transfection is generally one of the simplest and least cumbersome techniques, as most institutions have virus vector core facilities. Researchers can also make use of publicly available viral vectors deposited by scientists on Addgene.
- Most viruses can create stable transfection, which means that the genetic material is stably inserted and can be expressed over several generations of cells or across the lifespan of the cells.
- There is a limit to the size of the genetic materials that can be packaged inside the viruses. For instance, the maximum size of DNA is about 11 kbp for lentivirus, and there is an exponential decrease in packing efficiency if the limit is exceeded.
- Many viruses can cause host cells to lyse and die.
- A higher level of biological safety containment may be necessary due to the use of viruses.
This class of methods uses chemicals such as lipids and calcium phosphate for transfection. Lipid-mediated transfection, also known as lipofection, uses lipids with similar properties to that of cell membrane. The positively charged lipids associate with the negatively charged phosphate groups of genetic materials. The complex then fuses with cell membrane for delivery. It has been found that positively charged lipids also generate higher transfection efficiency due to better association with negatively charged cell membrane. The calcium phosphate method involves mixing DNA-calcium chloride mixture into phosphate solution to form precipitate. The precipitate is then uptake by cells via endocytosis.
- Success rates are high, although it can be cell-type dependent. For instance, this method works well for cells cultured on flat dishes and not in vivo environments.
- The reagents, such as lipofectamine, are readily available and inexpensive.
- Factors such as serum in media and cell confluency affect the transfection efficiency.
- For protocol using calcium phosphate, it is important to have consistency in reagent properties, such as pH, to avoid compromised efficiency.
- The calcium phosphate method does not work for cells grown in media with high phosphate levels, such as RPMI.
This class of methods utilizes physical means such as mechanical and electrical forces to induce transient opening of cell membrane for transfection. Microinjection is a method in which a glass pipette is used to manually inject genetic materials into each cell. Electroporation generates an electrical field across the cell membrane to induce pore opening. Genetic materials can then enter the cells when the pores are transiently open. Magneto-transfection and laser/opto-transfection are relatively new techniques that use magnetic forces/optical energy to permeabilize cells to deliver genetic material into them.
- Apart from micropipette injection, these methods can generally transfect many cells each time and be easily performed.
- These methods work better for adherent cells to create uniform electrical and magnetic fields for transfection.
- Setup using high-power lasers can be expensive.
Although methods for transfection are well-established, there are still unsolved problems and innovative research trying to overcome them. The Gradinaru lab at Caltech is investigating the efficiency of their viral vector libraries to cross the blood barrier, which will be very useful for drug delivery application. Chemists are also working to design smart polymer carriers sensitive to stimuli such as heat and pH for gene transfection. Advances in microfabrication have also facilitated innovative work using microfluidic platforms for better control in electroporation and micropipette injector array.
I hope this short overview of various transfection methods will be useful for your work!
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