Reporter Knockin Mouse Models

For academic research: Gene expression monitoring Cellular response monitoring 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

+	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

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.

Itziar Terradillos, Itziar Bonilla-Del Río, Nagore Puente, Maitane Serrano, Amaia Mimenza, Leire Lekunberri, Ilazki Anaut-Lusar, Leire Reguero, Inmaculada Gerrikagoitia, Samuel Ruiz de Martín Esteban, Cecilia J Hillard, María T Grande, Julián Romero, Izaskun Elezgarai, Pedro Grandes.
Altered glial expression of the cannabinoid 1 receptor in the subiculum of a mouse model of Alzheimer's disease.
Glia. 2023 Apr

Jun W Kim, Julie H Ko, Julien Sage.
DLL3 regulates Notch signaling in small cell lung cancer.
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Julia Leschik, Antonietta Gentile, Cigdem Cicek, Sophie Péron, Margaryta Tevosian, Annika Beer, Konstantin Radyushkin, Anna Bludau, Karl Ebner, Inga Neumann, Nicolas Singewald, Benedikt Berninger, Volkmar Lessmann, Beat Lutz.
Brain-derived neurotrophic factor expression in serotonergic neurons improves stress resilience and promotes adult hippocampal neurogenesis
Prog Neurobiol. 2022 Jul 21

Yu Ting Ong, Jorge Andrade, Max Armbruster, Chenyue Shi, Marco Castro, Ana S H Costa, Toshiya Sugino, Guy Eelen, Barbara Zimmermann, Kerstin Wilhelm, Joseph Lim, Shuichi Watanabe, Stefan Guenther, Andre Schneider, Francesca Zanconato, Manuel Kaulich, Duojia Pan, Thomas Braun, Holger Gerhardt, Alejo Efeyan, Peter Carmeliet, Stefano Piccolo, Ana Rita Grosso, Michael Potente.
A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth
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Inwoo Hwang, Byeong-Seong Kim, Hyo Rim Ko, Seongbong Cho, Ho Yun Lee, Sung-Woo Cho, Dongryeol Ryu, Sungbo Shim, Jee-Yin Ahn.
Cerebellar dysfunction and schizophrenia-like behavior in Ebp1-deficient mice.
Mol Psychiatry. 2022 Apr

Isabel Fernández-Pisonero, Laura Clavaín, Javier Robles-Valero, L Francisco Lorenzo-Martín, Rubén Caloto, Blanca Nieto, Carmen García-Macías, Clara L Oeste, Manuel Sánchez-Martín, Antonio Abad, Alejandro Hortal, Dolores Caballero, Marcos González, Mercedes Dosil, Balbino Alarcón, Xosé R Bustelo.
A hotspot mutation targeting the R-RAS2 GTPase acts as a potent oncogenic driver in a wide spectrum of tumors.
Cell Rep. 2022 Mar 15

Alejandro M Hortal, Clara L Oeste, Claudia Cifuentes, Miguel Alcoceba, Isabel Fernández-Pisonero, Laura Clavaín, Rut Tercero, Pilar Mendoza, Verónica Domínguez, Marta García-Flores, Belén Pintado, David Abia, Carmen García-Macías, Almudena Navarro-Bailón, Xosé R Bustelo, Marcos González, Balbino Alarcón.
Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia
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Charlotte Douglas, Valdone Maciulyte, Jasmin Zohren, Daniel M Snell, Shantha K Mahadevaiah, Obah A Ojarikre, Peter J I Ellis, James M A Turner
CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes
Nat Commun. 2021 Dec 3

A Fatehullah, Y Terakado, S Sagiraju, T L Tan, T Sheng, S H Tan, K Murakami, Y Swathi, N Ang, R Rajarethinam, T Ming 6, P Tan, B Lee, N Barker.
A tumour-resident Lgr5 + stem-cell-like pool drives the establishment and progression of advanced gastric cancers
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Li-Chiung Lin, Tezz Quon, Susanna Engberg, Amanda E Mackenzie, Andrew B Tobin, Graeme Milligan
G Protein-Coupled Receptor GPR35 Suppresses Lipid Accumulation in Hepatocytes.
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Noureddine Ben Khalaf, Wedad Al-Mashoor, Azhar Saeed, Wassim Raslan, Halla Bakheit, Ameera Abdulhadi, Ammar Marouani, Safa Taha, Moiz Bakhiet, M Dahmani Fathallah.
Knocking down Israa, the Zmiz1 intron-nested gene, unveils interrelated T cell activation functions in mouse.
Biochem Biophys Rep. 2021 Aug 9

Lisa Baker, Moses Tar, Adam H Kramer, Guillermo A Villegas, Rabab A Charafeddine, Olga Vafaeva, Parimala Nacharaju, Joel Friedman, Kelvin P Davies, David J Sharp.
Fidgetin-like 2 negatively regulates axonal growth and can be targeted to promote functional nerve regeneration.
JCI Insight. 2021 May 10

Sharon O Jensen-Cody, Kyle H Flippo, Kristin E Claflin, Yavuz Yavuz, Sarah A Sapouckey, Grant C Walters, Yuriy M Usachev, Deniz Atasoy, Matthew P Gillum, Matthew J Potthoff.
FGF21 Signals to Glutamatergic Neurons in the Ventromedial Hypothalamus to Suppress Carbohydrate Intake.
Cell Metab. 2020 Aug 4

Ravit Gabay Yehezkely, Shelly Zaffryar-Eilot, Anna Kaganovsky, Nurit Fainshtain Malka, Rohtem Aviram, Ido Livneh, Peleg Hasson.
Intracellular Role for the Matrix-Modifying Enzyme Lox in Regulating Transcription Factor Subcellular Localization and Activity in Muscle Regeneration.
Dev Cell. 2020 May 18.

Inwoo Hwang, Jee-Yin Ahn.
Dysregulation of Epigenetic Control Contributes to Schizophrenia-Like Behavior in Ebp1 +/- Mice.
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Martin Kolev, Erin E West, Natalia Kunz, Daniel Chauss, E Ashley Moseman, Jubayer Rahman, Tilo Freiwald, Maria L Balmer, Jonas Lötscher, Sarah Dimeloe, Elizabeth C Rosser, Lucy R Wedderburn, Katrin D Mayer-Barber, Andrea Bohrer, Paul Lavender, Andrew Cope, Luopin Wang, Mariana J Kaplan, Niki M Moutsopoulos, Dorian McGavern, Steven M Holland, Christoph Hess, Majid Kazemian, Behdad Afzali, Claudia Kemper.
Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.
Immunity. 2020 Mar 17.

Sophie J Bradley, Colin Molloy, Paulina Valuskova, Louis Dwomoh, Miriam Scarpa, Mario Rossi, Lisa Finlayson, Kjell A Svensson, Eyassu Chernet, Vanessa N Barth, Karolina Gherbi, David A Sykes, Caroline A Wilson, Rajendra Mistry, Patrick M Sexton, Arthur Christopoulos, Adrian J Mogg, Elizabeth M Rosethorne, Shuzo Sakata, R A John Challiss, Lisa M Broad, Andrew B Tobin.
Biased M1-muscarinic-receptor-mutant Mice Inform the Design of Next-Generation Drugs.
Nat Chem Biol. 2020 Mar.

Nakawesi J, This S, Hütter J, Boucard-Jourdin M, Barateau V, Muleta KG, Gooday LJ, Thomsen KF, López AG, Ulmert I, Poncet D, Malissen B, Greenberg H, Thaunat O, Defrance T, Paidassi H, Lahl K.
αvβ8 integrin-expression by BATF3-dependent dendritic cells facilitates early IgA responses to Rotavirus.
Mucosal Immunol. 2020 Mar 11.

Werner Y, Mass E, Ashok Kumar P, Ulas T, Händler K, Horne A, Klee K, Lupp A, Schütz D, Saaber F, Redecker C, Schultze JL, Geissmann F, Stumm R.
Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke.
Nat Neurosci. 2020 Mar.

Chen C, Sun MA, Warzecha C, Bachu M, Dey A, Wu T, Adams PD, Macfarlan T, Love P, Ozato K.
HIRA, a DiGeorge Syndrome Candidate Gene, Confers Proper Chromatin Accessibility on HSCs and Supports All Stages of Hematopoiesis.
Cell Rep. 2020 Feb 18.

Hyo Rim Ko, Inwoo Hwang, Eun-Ju Jin, Taegwan Yun, Dongryeol Ryu, Jong-Sun Kang, Kye Won Park, Joo-Ho Shin, Sung-Woo Cho, Kyung-Hoon Lee, Keqiang Ye, Jee-Yin Ahn.
Roles of ErbB3-binding Protein 1 (EBP1) in Embryonic Development and Gene-Silencing Control.
Proc Natl Acad Sci U S A. 2019 Dec 3.

Ben Khalaf N, Al-Mashoor W, Saeed A, Al-Mehatab D, Taha S, Bakhiet M, Fathallah MD.
The mouse intron-nested gene, Israa, is expressed in the lymphoid organs and involved in T-cell activation and signaling.
Mol Immunol. 2019 May 13.

Daniele Bolognini, Natasja Barki, Adrian J Butcher, Brian D Hudson, Eugenia Sergeev, Colin Molloy, Catherine E Moss, Sophie J Bradley, Christian Le Gouill, Michel Bouvier, Andrew B Tobin, Graeme Milligan.
Chemogenetics defines receptor-mediated functions of short chain free fatty acids.
Nat Chem Biol. 2019 May.

Amini SE, Gouyer V, Portal C, Gottrand F, Desseyn JL.
Muc5b is mainly expressed and sialylated in the nasal olfactory epithelium whereas Muc5ac is exclusively expressed and fucosylated in the nasal respiratory epithelium.
Histochem Cell Biol. 2019 Apr 27.

Xu W, Cherrier DE, Chea S, Vosshenrich C, Serafini N, Petit M, Liu P, Golub R, Di Santo JP.
An Id2RFP-Reporter Mouse Redefines Innate Lymphoid Cell Precursor Potentials.
Immunity. 2019 Apr 16.

Leschik J, Eckenstaler R, Endres T, Munsch T, Edelmann E, Richter K, Kobler O, Fischer KD, Zuschratter W, Brigadski T, Lutz B, Lessmann V.
Prominent Postsynaptic and Dendritic Exocytosis of Endogenous BDNF Vesicles in BDNF-GFP Knock-in Mice.
Mol Neurobiol. 2019 Mar 30.

Hung LY, Sen D, Oniskey TK, Katzen J, Cohen NA, Vaughan AE, Nieves W, Urisman A, Beers MF, Krummel MF, Herbert DR.
Macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a Trefoil factor 2-dependent mechanism.
Mucosal Immunol. 2018 Oct 18.

Lu L, Marisetty A, Liu B, Kamal MM, Gumin J, Veo B, Cai Y, Kassem DH, Weng C, Maynard ME, Hood KN, Fuller GN, Pan ZZ, Cykowski MD, Dash PK, Majumder S.
REST overexpression in mice causes deficits in spontaneous locomotion.
Sci Rep. 2018 Aug 14.

Vanden Bempt M, Demeyer S, Broux M, De Bie J, Bornschein S, Mentens N, Vandepoel R, Geerdens E, Radaelli E, Bornhauser BC, Kulozik AE, Meijerink JP, Bourquin JP, de Bock CE, Cools J.
Cooperative Enhancer Activation by TLX1 and STAT5 Drives Development of NUP214-ABL1/TLX1-Positive T Cell Acute Lymphoblastic Leukemia.
Cancer Cell. 2018 Aug 13.

López A, Aparicio N, Pazos MR, Grande MT, Barreda-Manso MA, Benito-Cuesta I, Vázquez C, Amores M, Ruiz-Pérez G, García-García E, Beatka M, Tolón RM, Dittel BN, Hillard CJ, Romero J.
Cannabinoid CB2 receptors in the mouse brain: relevance for Alzheimer's disease.
J Neuroinflammation. 2018 May 24.

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.