0:00-0:30 | What is transfection?
0:30-1:24 | Transient transfection explained
1:24-2:50 | Stable transfection explained
2:50-3:42 | Why is transfection useful?
Transfection is the process of artificially introducing nucleic acids such as DNA or RNA into cells, utilizing any means other than viral infection. This can cause the cell to change, making it possible to study gene function as well as protein expression in the context of the cell.
There are two main types of transfection, transient transfection and stable transfection. Transient transfection introduces a nucleic acid only for a limited time since it is not integrated into the genome of the cell. Therefore this genetic material will not be passed on during cell division, in other words, there is no cross generational inheritance. Depending on what method is utilized, a transgene transfected in a transient manner can usually be detected for 1-7 days. Supercoiled DNA is most efficient but siRNA, miRNA, mRNA and even proteins can also be used. Transfected DNA is translocated into the nucleus for transcription while the transfected RNA remains in the cytosol where it is expressed within minutes!
Stable transfection allows foreign DNA to either integrate into the cellular genome or otherwise remain as episomal plasmid. This allows the DNA to remain permanently, in other words, DNA that has been integrated through stable transfection is also inherited cross generationally. However, the expression level is usually lower in stable transfection compared to transient transfection since usually it is only possible to integrate a few copies of foreign DNA into the genome. Stable integration is also a lot rarer making effective gene delivery and the incorporation of a selective marker vital. Best ways to select successfully transfected cells is to include a selectable marker such as an antibiotic resistant gene or something similar. Then, one can expose cells to the substance that successfully transfected cells have become resistant to in order to sort out unsuccessful cases. In addition, one can use a marker that causes morphological or phenotypical changes. To successfully perform stable transfection, linear DNA is preferable to supercoiled DNA.
So how is transfection useful? Well it is most commonly used to express a protein of interest in cultured cells through the use of a plasmid vector or mRNA. One can also use transfection to express protein in eukaryotic cells, allowing for proper folding and post-translational modifications. In addition, transfection can also be used to create pluripotent stem cells (iPSC) and various therapeutic molecules. Finally transfection is also used as a way to cause gene inhibition by using RNA interference or RNAi.
25 июл 2024
