Humanized KI mouse

Case 1 | Study of a human-specific receptor

‍Schwarz F, et al. Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen. EMBO J. 2017.

E. coli K1 is a human-specific pathogen that appears to exploit a receptor Siglec-11 expressed only in the human brain.
Mice do not have paired Siglec receptors.

Model: Mice expressing a chimeric, human-type paired Siglec receptor E16.

Aim: To demonstrate that activating Siglecs confer better protection against bacterial infection in vivo.

Results: Siglec-E16 was able to produce protective inflammatory responses to bacterial infection.

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Figure 1. Schematic representation of Siglec-E and Siglec-E16 receptors.

The parts of Siglec-E16 derived from mouse Siglec-E or human Siglec-16 are drawn in gray and black, respectively.

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Figure 2. Activating Siglecs confer protection against E. coli K1 challenge.

A) Decreased E. coli survival in blood from E16/E16 mice, 1 hour after infection.

B) Less recovered bacteria from blood, spleen, and liver from E16/E16 mice.

C) Increased cytokine levels in serum from E16/E16 mice, 1 hour after bacterial challenge.

Case 2 | Biomarker identification in a rare disease ALD model

van de Beek MC, et al. C26:0-Carnitine Is a New Biomarker for X-Linked Adrenoleukodystrophy in Mice and Man. PLoS One. 2016.

X-linked adrenoleukodystrophy, ALD, is a progressive neurodegenerative disease, and a result of very-long-chain fatty acid (VLCFA) buildup caused by relevant enzymes not functioning properly (mutations in Abcd1).

Model: Abcd1 Knockout overexpressing human Evolv1, an essential enzyme in the elongation of VLCFA (C22:0 to C26:0).

Aim: To develop an ALD model with increased VLCFA (>C22) levels in the central nervous system for the identification of new biomarkers for ALD.

Results: The VLCFA C26:0-Carnitine is a new biomarker for ALD that reflects elevated VLCFA levels present in the central nervous system of mice and humans.

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Figure 1. C26:0-carnitine is highly elevated in central nervous tissue.

C26:0-carnitine levels in brain and spinal cord from wildtype (n=6), Abcd1^y/- knockout (n=6) and Abcd1^y/-;Cnp-ELOVL1^+/- (n=6) mice.

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Figure 2. C26:0-carnitine is highly elevated in mouse and human bloodspots.

Left) Bloodspot C26:0-carnitine in wildtype (n=8), Abcd1^y/-knockout (n=10) and Abcd1^y/-;Cnp-ELOVL1^+/- (n=6) mice.

Right) Bloodspot C26:0-carnitine in controls (n=23) and ALD patients (n=10).

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Case 3 | Functional validation of an antibody against GPCR

Butcher AJ, et al. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory. J Biol Chem. 2016.

Establishing the in vivo activation status of G protein-coupled receptors (GPCRs) would not only indicate physiological roles of GPCRs but would also aid drug discovery by establishing drug-receptor engagement.

Model: Mice expressing a humanized, mutated form of the GPCR M1mAChR.

Aim: To develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor in vitro and in vivo.

Results: Phosphorylation sites can be used to probe the activation status of GPCRs during physiological responses and on drug treatment.

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Figure 1. Detection of phosphorylation of M1 DREADD receptor in the hippocampus following receptor activation with a selective agonist.

A) Illustration of M1 DREADD receptor. Two point mutations were introduced into the human M1 mAChR, abolishing the activation by ACh, but instead the receptor could be activated by the drug CNO.

B) Fixed sections from M1 mAChRKO mice (M1-KO) or M1 DREADD KI mice treated with vehicle or CNO were co-stained with anti-HA (green) and anti phospho-specific antibodies (red).

The arrows indicate two neurons, where the staining for the receptor and the phosphorylated receptor occur in the same neuron.

The areas marked by the white box are magnified in the lower panels.

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Case 4 | Human disease model for acute myeloid leukemia (AML)

Sportoletti P, et al. The human NPM1 mutation A perturbs megakaryopoiesis in a conditional mouse model. Blood. 2013.

The NPM1 mutation is a frequent genetic alteration in acute myeloid leukemia (AML). Despite progress in the clinical and biological characterization of NPM1-mutated AML, the role of NPM1 mutation in leukemogenesis in vivo has not been fully elucidated.

Model: Mice that conditionally express the most common human NPM1 mutation (type A) in the hematopoietic compartment.

Aim: To develop an AML model to identify the role of NPM1 in leukemogenesis.

Results: The NPM1 mutant affects megakaryocytic development in mice and mimics some features of human NPM1-mutated AML.

Figure 1. Immature megakariocytes were increased 2-fold in heterozygous (Npm1-TCTG/WT;Cre1) and 4-fold in homozygous (Npm1-TCTG/TCTG;Cre1) mutant mice.

A) Flow cytometric analysis of single-cell suspension of BM.

B) Quantification of CD411 megakaryocytic cells in the BM of age-matched mutant mice analyzed as in panel A.

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Figure 2. Perturbation in megakaryocyte growth shown in in vitro colony-forming assays.

A) Npm1-TCTG/WT;Cre1 BM cells formed significantly more megakaryocytic colonies (CFU-MK) than Cre2 control in semisolid media (34.00 6 5.367 vs 9.0 6 1.265 CFUMK/ 105BMcells; n56, P,.01).

B) CFU-MK sizes were similar.