Let’s say you work with a gene, and it has wonderful potential. Excitedly, you throw your gene into the cells and voila! It’s there. Great. Now what?
Genes are powerful tools for directing cell activity, but thanks to that curiosity characteristic of scientists, we want to know more:
- We want to be able to confirm the integration of our gene and watch it in action
- We want to be able to select clones with our gene of interest
- We want to investigate the concerted effect of two genes working in conjunction
So what do you do? You had one. You studied it through and through. So now, naturally, you move to two.
Introducing the bicistronic plasmid
As the name suggests, bicistronic plasmids contain two distinct genes of interest within one vector. The vector transports the genes together into the cells, which means that every cell with one gene also has the other. On the contrary, cotransfection does not have such a guarantee, and you may encounter cells with only one of your genes. Therefore, transporting your genes together adds a new level of reliability for transfection efficiency.
Bicistronic plasmids have another distinct advantage: they open the door to a vast array of gene combinations. To advance research on your specific gene, you can add a selection gene or reporter genes. To extend your research to protein interaction, you can combine multiple genes and track their function with relation to each other.
Alright, you want a bicistronic plasmid. How do you link the genes? Allow me to introduce you to three methods for co-expressing multiple genes.
The most straightforward method for coexpression is to use a second promoter within the same vector. In the two-promoter system, your vector contains two separate expression cassettes with a different promoter for each gene.
Two-promoter systems are ideal when you want to have both proteins expressed at the same level because they tend to provide equal expression. However, the efficiency of expression can be affected by the size of the promoters and genes. Issues with this system arise when space is limited in the vector.
Internal ribosome entry site (IRES)
In this method, you hijack the native ability of a virus to express additional genes from within a stretch of mRNA (cap independent translation). IRES linkage works by transcribing both genes in one stretch of mRNA, and then translating the genes separately by initiating translation within the mRNA at the IRES.
Using IRES eliminates the need for a second promoter and thereby saves space, but there are some drawbacks. The second gene, driven by IRES, typically has lower expression than when it is expressed by itself, and the efficiency of IRES varies by cell type. It is also important to note that the IRES sequence can vary, which affects its expression ratio. IRES elements have been isolated from encephalomyocarditis virus and other picornaviruses, especially foot and mouth disease virus, and can vary in length. Some commercial versions also contain mutations.
Peptide 2A (P2A) sequences
These sequences also come from picornaviruses and have an interesting ability to self-cleave. P2A sequences sit in between your two genes of interest and cause ribosomal “skipping” during translation, which results in a missing peptide bond and effectively separates the two proteins. The major advantage of P2A is its size—just 19 amino acids. Another positive is that expression of both genes is comparable to normal expression levels.
There are two main obstacles when using P2A. First, both genes retain part of the P2A sequence as a result of the cleavage. A few amino acid residues from the tag are left on the upstream gene and a proline is left at the beginning of the second gene. This can affect the functionality or targeting of the proteins. Secondly, expression efficiency is dependent upon cleavage efficiency, which can be affected by the downstream gene. If cleavage doesn’t work, you have a fusion protein whose function decreases or ceases unless the genes can be expressed as a polyprotein.
To sum up, choosing the right method for co-expression depends on your specific needs and limitations. The two-promoter system is powerful and efficient as long as you have enough space within your vector. IRES is a useful alternative when space is limited and the expression ratio is not important. The P2A system opens opportunities for larger transgenes or even a third gene, but it may involve extra optimization.
Whichever you choose, bicistronic plasmids can add a versatile element to your research and provide an exciting potential for dynamic molecular biology.