A humanized Knockin mouse defines a model in which a mouse gene is replaced by either a human gene, genomic sequence or regulatory element.

This substitution can target parts of the gene, and subsequently specific domains of the protein, as well as certain mouse regulatory elements such as the promoter.

The human protein/domain is then expressed in cells and tissues where the mouse protein was expressed.

Applications

For academic research:

  • Phenocopy a human disease to decipher causes and molecular mechanisms
  • Create a mouse model that is tolerant of human antigen to study human peptide functions

For bio-pharmaceutical research & development:

  • Phenocopy human diseases for drug development and safety studies
  • Provides a model to study efficacy when endogenous mouse target shows low affinity to xenobiotics or biologics
  • Create a mouse model tolerant of human antigen for antibody or antibody drug conjugate (ADC) testing
  • Human cytokine expression to improve human immune system engraftment

Strengths of humanized Knockin mouse models

  • Physiological expression patterns due to the murine promoter and regulatory elements
  • Murine gene is inactivated; no hybrid sequences (like, e.g., in transgenic animals)
  • Combination with access to Knockout model for specificity studies

Limitations of humanized Knockin mouse models

  • Risk of differential expression in mouse vs. human, due to differences in protein regulation
  • Low conservation between human/mouse protein => risk of hypomorphism up to protein Knockout
    →  Limitation can be bypassed by using chimeric molecules to improve functionality in mouse cells

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.

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.

Figure 1 - Siglec-E16 mice

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

Figure 2a - Siglec-E16 mice

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.

Figure 2bc - Siglec-E16 mice

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.

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), Abcd1y/- knockout (n=6) and Abcd1y/-;Cnp-ELOVL1+/- (n=6) mice.

Figure 1 - Abcd1 mice

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

Left) Bloodspot C26:0-carnitine in wildtype (n=8), Abcd1y/-knockout (n=10) and Abcd1y/-;Cnp-ELOVL1+/- (n=6) mice.

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

Figure 2 - Abcd1 mice

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.

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.

Figure 1a - GPCR M1mAChR mice

Figure 1b - GPCR M1mAChR mice

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.

Figure 1a - NPM1 mice

Figure 1b - NPM1 mice

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.

Figure 2ab - NPM1 mice
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