Reporter KI mouse

Case Studies

The reporter allows the researcher to quantify the gene expression level, track the cells expressing the gene of interest, and monitor the regulation of the gene. Historically, such models have been generated, for example, to quantify the effector function of regulatory T cells (case 2), to quantify cytokine production, to follow cytokine-producing cells temporally, or monitor rapidly dividing erythroid progenitors (case 1).

Both model cases use IRES to insert the reporter gene at the 3’ UTR of the gene of interest. Upon activation, the mRNA comprised of the construct IRES-reporter-target gene, is translated as two independent proteins.

Case 1 | IRES-luciferase model for in vivo monitoring of hematopoietic stem cells and rapidly dividing erythrocyte precursors (created by genOway)

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 to luciferase expression.

Case 2 | Foxp3-IRES-mRFP (FIR) reporter mouse model to monitor regulatory T cell activity

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 U S A. 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) (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 genotyping 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.