Reporter Knockin Mouse Models

 



Get supplemental information, a quote, and estimated timeframe for generating your reporter Knockin mouse.

 

A reporter Knockin mouse defines an animal model in which fluorescent, bioluminescent proteins or biochemical tags are inserted into the genome. The reporter can replace a gene, be fused to a protein or inserted into the 3' UTR.

It is a particularly useful model when there are no available antibodies for the target protein.

Infographic: Reporter Knockin mouse model

Typical applications for reporter Knockin mouse models


For academic research:
  • Gene expression monitoring
  • Cellular response montoring
  • Knockout confirmation readout
  • Protein, cell and cell lineage trafficking
  • Cell sorting
  • Biochemical analysis, e.g., protein-protein interaction
For bio-pharmaceutical research & development:
  • Readout of responses to specific stimuli
  • Drug screening
  • In vivo sensing
  • Biomarkers

Strengths and limitations of reporter Knockin mouse models

+
  • In vivo monitoring of desired events without using antibodies
  • Usable in all kind of models (Knockouts, Knockins, humanizations)
  • Reporter can be fused to promoters, transcripts or via linkers to avoid disrupting the function of the target protein
-
  • Fusing the reporter to a protein may alter protein conformation, localization and functionality
    →  Limitation can be bypassed by applying IRES co-expression technology
  • 1. 3' UTR approaches are not quantitative
    2. Essential to preserve all regulatory elements to keep expression level and pattern
    3. Challenging when multiple isoforms have been described
    → Must be anticipated/integrated in a profound scientific risk assessment prior to model development

 


Case studies and publications on our reporter Knockin mouse models

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.

Publications

Zhang Z, Newton K, Kummerfeld SK, Webster J, Kirkpatrick DS, Phu L, Eastham-Anderson J, Liu J, Lee WP, Wu J, Li H, Junttila MR, Dixit VM.
Transcription factor Etv5 is essential for the maintenance of alveolar type II cells.
Proc Natl Acad Sci U S A. 2017 Apr 11.

Portal C, Gouyer V, Gottrand F, Desseyn JL.
Preclinical mouse model to monitor live Muc5b-producing conjunctival goblet cell density under pharmacological treatments.
PLoS One. 2017 Mar 29.

Koechlein CS, Harris JR, Lee TK, Weeks J, Fox RG, Zimdahl B, Ito T, Blevins A, Jung SH, Chute JP, Chourasia A, Covert MW, Reya T.
High-resolution imaging and computational analysis of haematopoietic cell dynamics in vivo.
Nat Commun. 2016 Jul 18.

Nakamura Y, Morrow DH, Modgil A, Huyghe D, Deeb TZ, Lumb MJ, Davies PA, Moss SJ.
Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse.
J Biol Chem. 2016 Jun 3.

Butcher AJ, Bradley SJ, Prihandoko R, Brooke SM, Mogg A, Bourgognon JM, Macedo-Hatch T, Edwards JM, Bottrill AR, Challiss RA, Broad LM, Felder CC, Tobin AB.
An antibody biosensor establishes the activation of the M1 muscarinic acetylcholine receptor during learning and memory.
J Biol Chem. 2016 Jan 29.

Iqbal AJ, McNeill E, Kapellos TS, Regan-Komito D, Norman S, Burd S, Smart N, Machemer DE, Stylianou E, McShane H, Channon KM, Chawla A, Greaves DR
Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.
Blood. 2014 Oct 9.

Shih PY, Engle SE, Oh G, Deshpande P, Puskar NL, Lester HA, Drenan RM
Differential expression and function of nicotinic acetylcholine receptors in subdivisions of medial habenula.
J Neurosci. 2014 Jul 16.

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.

Foronda M, Martínez P, Schoeftner S, Gómez-López G, Schneider R, Flores JM, Pisano DG, Blasco MA
Sox4 Links Tumor Suppression to Accelerated Aging in Mice by Modulating Stem Cell Activation.
Cell Rep. 2014 Jul 15.

Thomas PS, Rajderkar S, Lane J, Mishina Y, Kaartinen V
AcvR1-mediated BMP signaling in second heart field is required for arterial pole development: Implications for myocardial differentiation and regional identity.
Dev Biol. 2014 Jun 15.

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 Mar 25.

Winkelmann A, Maggio N, Eller J, Caliskan G, Semtner M, Häussler U, Jüttner R, Dugladze T, Smolinsky B, Kowalczyk S, Chronowska E, Schwarz G, Rathjen FG, Rechavi G, Haas CA, Kulik A, Gloveli T, Heinemann U, Meier JC.
Changes in neural network homeostasis trigger neuropsychiatric symptoms.
J Clin Invest. 2014 Jan 16.

Imudia AN, Wang N, Tanaka Y, White YA, Woods DC, Tilly JL.
Comparative gene expression profiling of adult mouse ovary-derived oogonial stem cells supports a distinct cellular identity.
Fertil Steril. 2013 Nov.

Dart DA, Waxman J, Aboagye EO, Bevan CL.
Visualising androgen receptor activity in male and female mice.
PLoS One. 2013 Aug 7.

Zhang J, Twelvetrees AE, Lazarus JE, Blasier KR, Yao X, Inamdar NA, Holzbaur EL, Pfister KK, Xiang X.
Establishing a novel knock-in mouse line for studying neuronal cytoplasmic dynein under normal and pathologic conditions.
Cytoskeleton. 2013 Mar 8<./em>

Haymaker CL, Guloglu FB, Cascio JA, Hardaway JC, Dhakal M, Wan X, Hoeman CM, Zaghouani S, Rowland LM, Tartar DM, VanMorlan AM, Zaghouani H.
Bone marrow-derived IL-13Rα,1-positive thymic progenitors are restricted to the myeloid lineage.
J Immunol. 2012 Apr 1.

Zhao T, Zhou X, Szabó N, Leitges M, Alvarez-Bolado G.
Foxb1-driven Cre expression in somites and the neuroepithelium of diencephalon, brainstem, and spinal cord.
Genesis. 2007 Dec.

Follow these links if you are looking for another Knockin mouse model or customized mouse model.