In a sentence: An IRES is an RNA sequence that forms a complex secondary structure that allows the initiation of translation from any position within an mRNA immediately downstream from where the IRES is located.
In order to explain what an IRES is we must first consider their origin and natural function. The most commonly used IRES sequences used in protein expression are derived from the picornavirus family of viruses, which include polioviruses and foot and mouth disease viruses. When these viruses enter cells they are often inflammatory and can induce the expression of genes that will prevent virus gene expression such as interferons. They also, as most viruses do, want to convert the cell into factories for producing their own proteins.
In order to this efficiently, these viruses express proteins that prevent ribosomes from engaging on mRNA molecules inside infected cells. This means that almost all protein production is stopped inside infected cells. However, the virus still needs to produce its own proteins and as such has evolved an RNA sequence that folds in a particular way that allows ribosomes to bind and start protein translation, independent of normal translation routes. This allows the virus to produce its proteins even though all 5’ cap-dependent translation has been inhibited within the cell. This also means that the virus is able to load ribosomes onto an mRNA from any region within an mRNA where the IRES is located. Hence why they are termed ‘internal’ ribosome entry sites.
For research, it is often desirable to express more than one gene from a stretch of DNA. The difficulty of predicting splice sequences, which would provide the ideal solution to this problem, has traditionally been made this hard to achieve. The observation that some viruses possess sequences that allow the loading of ribosomes for translation from ‘internal’ positions within an mRNA provided a potential solution. By positioning one coding sequence downstream of the 5’ cap/5’UTR in an mRNA, and a second gene downstream of an IRES sequence it is possible to allow the expression of two genes from a single mRNA (see figure below).
The main IRES sequences used for the expression of exogenous genes are derived from Foot and Mouth Disease virus (FMDV) and Encephalomyocarditis virus (EMCV). The EMCV virus sequence is much more frequently used and consistently delivers slightly higher expression in the cell types we have tested. However, the EMCV virus sequence is longer than the FMDV virus sequence, which can be an important consideration in space constraint expression systems (lentivirus and adenoviruses for example).
There is no shortage on literature pertaining to IRES expression levels. At Oxford Genetics we have spent more time on trying to understand IRES expression than almost any other sequence group. This is because they often show low expression levels compared to the upstream genes, and the level of expression varies from cell type to cell type. The following is a very brief conclusion of our findings:
Our current hypotheses and a possible explanation for the variable data in the literature are:
In our studies, using a CMV promoter, the EMCV IRES produces between 10 and 20 fold less protein than the upstream gene, whilst FMDV IRES produces typically 20-30 fold less.
Bacterial Origin Cloning: We currently have four alternative origins of replication for use in bacteria. Our bacterial origins of replication are normally flanked by two SwaI restriction sites. They will function if they are inserted in either orientation between these two sites.
Phage and Mammalian Origin Cloning: We also have a range of other origins of replication, including the SV40 origin of replication for mammalian cells and the F1 phage origin of replication that can be used to create single stranded DNA. These are currently available in either the SbfI or PacI sites respectively, however, they can be inserted into other positions on request.
We have produced three different types of plasmids containing industry standard transcription termination signals. These include:
The reason that we stock plasmids that only contain one terminator is because sometimes, despite the increased versatility, researchers may not want unnecessary terminators in their plasmids, for example when size constraints are an issue.
Remember that, while our plasmids are designed for easy cloning modifications, we are happy to do it for you if you prefer to outsource the cloning work.