Plasmids 101: Multicistronic Vectors

Co-expression of multiple genes is valuable in many experimental settings. To achieve this, scientists use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple or bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. We've discussed promoters before so in this blog post we’ll cover basics of multicistronic vectors: why they are useful, how they work, and how to get started with them.

Why Use multicistronic vectors?

Detecting cells that are expressing your gene, especially if you are studying a novel gene, is not always a straightforward process. Rather than try to directly detect your gene of interest, scientists have instead developed novel methods to co-express your gene along with a reporter, such as a fluorescence gene or a resistance gene. These reporters allow you to easily screen or select for cells that are expressing your gene of interest at high levels. Unlike vectors that express screenable or selectable markers from a unique promoter, multicistronic plasmids ensure that any cells that are positive for your marker should also be expressing your gene as they are both derived from the same transcript.

Of course multicistronic vectors do not have to exclusively be used as a means of detection; they are useful almost anytime you want to express multiple genes in the same cell. Although it is possible to drive co-expression by using a plasmid with multiple, individual expression cassettes, having the genes expressed from the same cassette is sometimes advantageous, particularly when only a portion of the plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is important.

How do multicistronic vectors work?

Scientists have "borrowed" some tricks discovered in positive single-stranded RNA viruses to allow for the efficient translation of multiple genes from a single transcript. The two strategies most widely incorporated into plasmids for research purposes are described below.

IRES Elements

Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event occurs for each mRNA. However, some bicistronic vectors take advantage of an element called an Internal Ribosome Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA.

In the figure above, you can see that the IRES element acts as another ribosome recruitment site, thereby resulting in co-expression of two proteins from a single mRNA.

IRES was originally discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic cells.^1,2 Since then, a variety of IRES sequences have been discovered - many from viruses, but also some from cellular mRNAs. What they all have in common is the ability to spark translation initiation independent of the 5’ cap. 

IRES elements are very useful and commonly found in bicistronic vectors; however, they do have some disadvantages. These elements are quite large (500-600 bp) and may take up precious space in viral transfer vectors with limited packaging capacity. Additionally, it may not be feasible to express more than two genes at a time using IRES elements. Further, scientists have reported lower expression of the downstream cistron due to factors such as the experimental cell type and the specific genes cloned into the vector.³ 

2A Peptides

To overcome some of the disadvantages of the IRES element, scientists have adapted "self-cleaving" 2A peptides into their muliticistronic vectors. These peptides, first discovered in picornaviruses, are short (about 20 amino acids) and produce equimolar levels of mulitple genes from the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.⁴ The "cleavage" occurs between the Glycine and Proline residues found on the C-terminus meaning the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the Proline. 

The table below lists the four common 2A peptides employed by scientists. 2A cleavage is universal in eukaryotic cells, and, although some scientists report close to 100% cleavage with some of these peptides, no consensus has been reached on which peptide works best. Likely the choice of specific 2A peptide will ultimately depend on a number of factors such as cell type or experimental conditions. 

Peptide	Amino acid sequence*
T2A:	(GSG) E G R G S L L T C G D V E E N P G P
P2A:	(GSG) A T N F S L L K Q A G D V E E N P G P
E2A:	(GSG) Q C T N Y A L L K L A G D V E S N P G P
F2A:	(GSG) V K Q T L N F D L L K L A G D V E S N P G P

* (GSG) residues can be added to the 5' end of the peptide to improve cleavage efficiency.

How do I get started?

If you are looking to co-express your gene of interest along with a fluorescent protein or selectable marker, it is easiest to start with a plasmid that already has the multicistronic element and reporter cloned in. In these plasmids you would simply clone your gene of interest into the multiple cloning site up or down stream of the IRES or 2A element (depending on the placement of the reporter gene).

Addgene's collection offers a variety of plasmids to express two or more genes, some of which are listed below. We should note that these vectors are primarily designed for bicistronic expression; however, many could be easily manipulated to express more than two genes.

Plasmid Name	Multicistronic Element	Expression Type
MSCV-IRES-EGFP	IRES	Retroviral
pMSCV-pBabeMCS-IRES-RFP	IRES	Retroviral
pMSCV-IRES-YFP II	IRES	Retroviral
pCMMP-MCS-IRES-Puro	IRES	Retroviral
pEF1a-IRES-Neo	IRES	Mammalian
MSCV-IRES-Luciferase	IRES	Retroviral
pWPI	IRES	Lentiviral
AmCyan-P2A-mCherry	P2A	Mammalian
pC5Kan-P2A	P2A	Insect
pUltra	P2A and T2A	Lentiviral
Ac5-STABLE2-neo	T2A	Insect

For the co-expression of multiple unique genes, you can start with a plasmid that has multiple cloning sites flanking the multicistronic element(s), or you could replace one of the reporter genes above with your gene or genes of interest. Some of the plasmids listed in the table above (and their related plasmids) are designed to have one or more of the genes replaced. 

Additionally, 2A peptides could be PCR-cloned between your genes of interest and you can then insert the whole multicistronic cassette into a backbone as a single unit. Although it is recommended to use the 2A peptides instead of an IRES when stoichiometrically equivalent levels of expression are required, we should also note that IRES and 2A peptides are not mutually exclusive elements. Labs have successfully utilized 2A and IRES elements within the same multicistronic vector, effectively making a construct that expresses multiple unique genes at equivalent levels upstream of an IRES fluorescent reporter for easy detection.⁵ 

Just released: Addgene's Plasmids 101 eBook

And now for some exciting news... You can now download Addgene's new eBook! Our goal was to create a one-stop reference guide for plasmids. We've combined our Plasmids 101 blog posts and additional resources into one downloadable PDF that you can save on your desktop. The Plasmids 101 eBook is designed to educate all levels of scientists and plasmid lovers and serves as an introduction to plasmids, allowing you to spend less time researching plasmid basic features and spend more time developing the clever experiments and innovative solutions necessary for advancing the field.

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References:

1. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Pelletier et al (Nature. 1988 Jul 28;334(6180):320-5.) PubMed.

2. A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. Jang et al. (J Virol. 1988 Aug;62(8):2636-43.) PubMed.

3. Highly Efficient Multicistronic Lentiviral Vectorswith Peptide 2A Sequences. Ibrahimi et al. (Hum Gene Ther. 2009 Aug;20(8):845-60. doi: 10.1089/hum.2008.188.) PubMed.

4. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim et al (PLoS One. 2011;6(4):e18556. doi: 10.1371/journal.pone.0018556. Epub 2011 Apr 29.) PubMed.

5. Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity. Holst et al (Nat Immunol. 2008 Jun;9(6):658-66. doi: 10.1038/ni.1611. Epub 2008 May 11.) PubMed.

Read more Plasmids 101 posts or browse Addgene's Plasmid Guide for more molecular biology and cloning information.

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