IRES: Internal Ribosome Entry Site
Generation of co-expression and/or reporter models
The IRES (internal ribosome entry site) allows the researcher to co-express several genes under the control of the same promoter. Within the past decade, the IRES has been used to develop hundreds of genetically modified models, and is considered to be the "go-to" technology for transgene co-expression in rodent animal models.
How does it work?
The endogenous promoter of the target gene directs the transcription of a single mRNA containing coding regions for the gene of interest (see figure below, blue) and the fluorescent reporter gene (see figure below, red). The reporter and the gene of interest are expressed as two independent proteins (non-fused), which ultimately allows the reporter protein to remain fully functional. The endogenous expression of the gene of interest is preserved as much as possible.
Applications of IRES technology
1) Generation of multigenic-based disease models
The IRES has been successfully used to generate models that mimic various human pathologies. Several cDNAs can be expressed under the control of the same promoter. This results in the co-expression of these genes in the same cells or tissues.
Case study: Advanced mouse model of Alzheimer's disease
Ryan D, Koss D, Porcu E, Woodcock H, Robinson L, Platt B, Riedel G.
Spatial learning impairments in PLB1Triple knock-in Alzheimer mice are task-specific and age-dependent.
Cell Mol Life Sci. 2013.
Platt B, Drever B, Koss D, Stoppelkamp S, Jyoti A, Plano A, Utan A, Merrick G, Ryan D, Melis V, Wan H, Mingarelli M, Porcu E, Scrocchi L, Welch A, Riedel G.
Abnormal Cognition, Sleep, EEG and Brain Metabolism in a Novel Knock-In Alzheimer Mouse, PLB1.
PLB1 gene construct design and expression levels
A) Genomic sequence of the transgene construct with the CaMKIIα promoter, human APP (hAPP), an internal ribosome entry site (IRES), followed by Tau (hTau); mutation sites are also indicated. hAPP and hTau were flanked by loxP and FRT sites, respectively. Neo: Neomycin selection cassette.
B) The mRNA expression of hAPP (left) and hTau (right) transgenes determined in the cortex was stable over time; homozygous (homo) animals showed 2–3-fold higher expression levels cf. heterozygous (het) mice.
2) Generation of reporter models
The IRES can be utilized to insert a reporter gene at the 3’ UTR of the gene of interest. Upon activation, the mRNA comprising the construct IRES-reporter-target gene, is translated as two independent proteins.
The reporter allows the researcher to quantify the gene expression level, to track the cells expressing the gene of interest, and to monitor the regulation of the gene. Historically, such models have been generated, for example, to quantify the effector function of regulatory T cells (see case 1 below), to quantify cytokine production, to follow cytokine-producing cells temporally, and to visualize a particular odorant receptor in olfactory neurons (see case 2 below).
Case studies: Luciferase and mRFP reporter
Case 1 | IRES-luciferase model for in vivo monitoring of hematopoietic stem cells and rapidly dividing erythrocyte precursors
Alvarez S, Díaz M, Flach J, Rodriguez-Acebes S, López-Contreras AJ, Martínez D, Cañamero M, Fernández-Capetillo O, Isern J, Passegué E, Méndez J.
Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality.
Nat Commun. 2015.
Mouse strain carrying a hypomorphic Mcm3 allele.
A) A modified mouse Mcm3 allele was designed with loxP sites flanking exons 14–17 and a luciferase reporter inserted at the 3' UTR under the control of an IRES element. The resultant allele (Mcm3-lox) was intended as a conditional KO, as Mcm3 expression could be ablated with Cre recombinase.
B) Expression of Mcm3-lox could be monitored by the bioluminescence activity associated with luciferase expression.
Case 2 | Foxp3-IRES-mRFP (FIR) reporter mouse model to monitor regulatory T cell activity (genOway's off-the-shelf model)
Dioszeghy V, Mondoulet L, Dhelft V, Ligouis M, Puteaux E, Dupont C, Benhamou PH.
The regulatory T cells induction by epicutaneous immunotherapy is sustained and mediates long-term protection from eosinophilic disorders in peanut-sensitized mice.
Clin Exp Allergy. 2014.
Wan YY, Flavell RA.
Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter.
Proc Natl Acad Sci USA. 2005.
Figure 1. Targeting IRES-mRFP reporter into the mouse Foxp3 locus.
A) Maps for mouse Foxp3 locus, targeting DNA construct, and the targeted Foxp3 locus. An 11-kb mouse genomic DNA, including exon 13 of Foxp3 gene, was excised by using BstZ17I (B) and HpaI (H) (Figure 1, top) and cloned into pEasy-Flox vector adjacent to the thymindine kinase (TK) selection marker. A cassette containing IRES-mRFP and loxP-flanked neomycin (Neo) selection marker was inserted into an SspI (S) site between the translation stop codon (UGA) and the polyadenylation signal (A2UA3) of Foxp3 gene (middle). A correctly targeted ES cell was used to create chimeras and germ-line-transmitted mice. The Neo gene was removed in vivo by using deletor mice transgenic for Cre recombinase to generate mice bearing targeted Foxp3 locus (lower).
B) PCR geno-typing FIR mice. Three primers (P1 to P3 as indicated) were designed to genotype FIR mice. PCR yielded 517-bp product for the wildtype (wt) Foxp3 allele and 470-bp product for targeted Foxp3 allele.
Figure 2. mRFP expression faithfully marks Foxp3-expressing CD4 T cells without compromising their regulatory activity, and Foxp3 expression was detected in different lymphocyte compartments.
Peripheral lymphocytes from FIR mice were harvested and stained with fluorophore-conjugated anti-CD4 and anti-CD25 antibodies. mRFP expression in CD4 T cells was monitored by flow cytometry (left). RNA was extracted from different populations of peripheral CD4 T cells (as indicated) purified from FIR mice by FACS. Relative mRNA levels of Foxp3 were determined by TaqMan real-time quantitative PCR, and the combined results of two experiments were plotted.
3) Generation of cell lineage tracking models
IRES insertion at the 3' UTR of the gene of interest allows us to develop models for cell lineage tracking. Researchers may use these models to investigate and analyze the spatiotemporal cell fate of given cell populations.
Case study: Intestinal epithelium lineage-tracing experiments
Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H.
Identification of stem cells in small intestine and colon by marker gene Lgr5.
Figure 1. Generation of mice expressing EGFP and Cre-ERt2 from a single bicistronic message by gene knock-in into the first exon of Lgr5.
Figure 2. Lineage tracing in the small intestine and colon.
A) Lgr5-eGFP-IRES-CreERt2 Knockin mouse crossed with Rosa26-lacZ reporter mice 12 hours after TAM injection.
B) Frequency at which the blue cells appeared at specific positions relative to the crypt bottom, according to the scheme in the inset. Most of the Cre1 LacZ-labeled crypt base columnar (CBC) cells occurred at positions between the Paneth cells, whereas only 10% of these cells were observed at the 14 position directly above the cells (blue line). Quantitative data on the position of long-term DNA-label-retaining cells obtained in adult mice after irradiation (marking the "14" intestinal stem cell) were published recently (Potten et al. J. Cell Sci. 2002). The graph shows a comparison of these data (red line) with the position of CBC cells carrying activated Cre.
C-E) Histological analysis of LacZ activity in small intestine one day after induction (C), five days after induction (D) and 60 days after induction (E).
F-H) Double-labeling of LacZ-stained intestine using periodic-acid–Schiff (PAS) demonstrates the presence of goblet cells (F, white arrows) and Paneth cells (G, blue arrows) in induced blue clones. Double-labeling with synaptophysin demonstrates the presence of enteroendocrine cells within the induced blue clones (H, black arrows).
I-K) Histological analysis of LacZ activity in colon one day after induction (I), five days after induction (J) and 60 days after induction (K).
4) Monitoring cell expression patterns in KO models
IRES systems are widely used in conditional Knockout mouse models developed by high throughput Knockout consortia such as KOMP, EUCOMM, and NorCOMM.
These systems allow the researcher to monitor gene expression patterns and gene regulation in various tissues and cells.
Ready-to-use Knockout models for in vivo studies
genOway provides access to more than 10,000 ready-to-use mouse models from EUCOMM. Among those, 1200 phenotyped lines have been aligned to human diseases related to immunology and inflammation, immuno-oncology, metabolism, neuroscience, pain, and cardiovascular- and musculoskeletal-based diseases.
EUCOMM vector map example:
5) Generation of tissue-specific Cre driver lines
The IRES is often used for the generation of Knockin Cre driver lines. The Knockin approach allows the driver line to faithfully mirror the expression pattern of the selected Cre expression locus.
The IRES sequence preserves expression of the target gene or allows the co-expression of a reporter gene. The reporter can provide information on expression level or on the expression pattern of Cre recombinase.
Case studies: Targeted recombination in neurons, and selective targeting with HDC-Cre
Case 1 | Demonstration of targeted recombination in PrRP neurons
Dodd GT, Worth AA, Nunn N, Korpal AK, Bechtold DA, Allison MB, Myers MG Jr, Statnick MA, Luckman SM.
The thermogenic effect of leptin is dependent on a distinct population of prolactin-releasing peptide neurons in the dorsomedial hypothalamus.
Cell Metab. 2014.
Cre recombinase is expressed exclusively in PrRP neurons
Dual-label immunostaining for PrRP (blue) and eGFP (red), as well as endogenous eGFP fluorescence (green), in the dorsomedial hypothalamic nucleus (DMH), nucleus of the tractus solitaries (NTS) and ventrolateral medulla (VLM) of PrRP-cre::eGFP mice.
Case 2 | Cell type selective genetic targeting with HDC-Cre mice
Zecharia AY, Yu X, Götz T, Ye Z, Carr DR, Wulff P, Bettler B, Vyssotski AL, Brickley SG, Franks NP, Wisden W.
GABAergic inhibition of histaminergic neurons regulates active waking but not the sleep-wake switch or PPF-induced loss of consciousness.
J Neurosci. 2012.
Gene-targeting strategy to produce the HDC-Cre Knockin mouse line.
The IRES-Cre cassette was placed into exon 12 of the hdc gene, between the stop (TAG) codon and the polyadenylation (pA) signal. An FRT-flanked neomycin resistance gene was inserted downstream for positive selection in ES cells. For negative selection, we used a GFP-expressing gene, which flanked the 3 homology arm, and analyzed nonfluorescent colonies. After successful targeting, the neomycin cassette (Neo) was removed by FLP recombinase to give the final targeted allele.