A protein function Knockout mouse defines an animal model in which one or more nucleotides are mutated in a way that the protein looses its function.

The insertion, deletion, nonsense and sense mutations alter the amino acid sequence of the given protein, affecting its function.


For academic research:

  • Study protein loss of function
  • Analyze the role of non-coding regions and regulatory elements
  • Investigate disease-causing loss-of-function mutations

For bio-pharmaceutical research & development:

  • Study drug-resistant mutants
  • Gene function study (function or expression)
  • Alter drug-antibody affinities
  • Pharmacological off-target and efficacy studies
  • Mimic human genetic diseases
  • Specificity studies

Strengths of protein function Knockout mouse models

  • Best way to reproduce human disease when due to mutations
  • High physiological relevancy of the scientific data obtained from the model (regulatory elements conserved, under control of endogenous promoter, expression of all splice variants, etc.) = cleaner way than classical Knockouts where the whole gene is deleted
  • Phenotype due only to the mutation: inactivation of a single function without disturbing other domains of the protein (also less risk of compensatory effects)

Limitations of protein function Knockout mouse models

  • Mutation of the gene of interest may affect development, resulting in an impaired phenotype or embryonic death
    → Limitation can be bypassed by applying conditions such as time-specific mutation activation
  • 1. Modification or disruption of splicing regulation
    2. Genetic redundancy
    → Can be assessed via constitutive Knockout of the gene of interest

Case studies

Case 1) Loss of protease activity: Model to overcome lack of suitable protease inhibitors

Yu JW, et al. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses. PLoS One. 2015.

MALT1 is part of signalosomes that play important roles in antigen receptor signaling. It functions as a scaffolding protein and as a protease to cleave and inactivate downstream inhibitory signaling proteins.
The study of these two distinct MALT1 activities is hampered by the lack of selective MALT1 protease inhibitors with suitable pharmacologic properties.

Model: Mice homozygous for the Malt1C472A, a catalytically inactive mutation inserted in the protease active site, leading to protease-dead Knockout.

Aim: Examine the role of the MALT1 protease activity in immune cells.

Results: MALT1 protease activity proved essential for the development of innate-like B cell populations. Further, Ig-responses to immunization with both T-dependent and T-independent antigens were dependent upon MALT1 protease activity.

Figure 1. A C472A mutation in MALT1 inactivates protease activity without changing protein expression.

Figure 1a - Malt1AC472A mice

A) Homologous recombination of the wt Malt1 locus (I) in C57BL/6-derived embryonic stem cells was used to introduce a catalytically inactive C472A mutation into exon 12 of the Malt1 gene.

The resulting Neo cassette-containing mice (II) were crossed to a FLP recombinase-expressing deleter mouse strain to generate animals carrying the protease-dead allele (III) used in these studies. These mice were in turn crossed to a Cre recombinase-expressing delete strain to excise exon 12 and generate a null allele (IV).

Close triangles, FRT sites; open triangles, loxP sites.

B) Purified total B cells from the spleens of wt, Malt1-/-, and Malt1C472A mice were treated with (+) or without (-) PMA plus ionomycin for 1 h and assessed by Western blotting for expression of MALT1, CYLD, Bcl10, and proteolytically cleaved CYLD and Bcl10.

Figure 2. The development of marginal zone (MZ) and B1 B cell populations is significantly impaired in Malt1PD/PD protease-dead mice.

Representative flow cytometry analysis of lymphocyte populations in MALT1 wt, Malt1-/-, and Malt1PD/PD mice.

Figure 2 - Malt1AC472A mice

A, C) MZ B cell (CD21-CD23+) population in the spleen

B, D) B1 B cell (IgMhiCD5lo) population in peritoneal fluid

Figure 3. In vivo antibody responses of wt, Malt1-/-, and Malt1PD/PD mice to immunization with T cell-dependent and -independent antigens.

MALT1 protease activity is required for maximal T-dependent and T-independent antibody responses in vivo.

Figure 3 - Malt1AC472A mice

A) When wt, Malt1-/-, and Malt1PD/PD were immunized with the T-dependent antigen KLH, wt mice produced high titers of anti-KLH IgM from days 7 through 28, and the expected Ig class switch occurred by day 14, with the anti-KLH IgG concentration increasing through day 28. Malt1PD/PD mice also displayed poor IgM and IgG responses to KLH immunization.

B) Immunization of wt, Malt1-/-, and Malt1PD/PD mice with the T-independent type II antigen TNP-Ficoll yielded a similar IgM profile to what was observed for T-dependent immunizations, however, the IgG response of Malt1PD/PD mice was not significantly affected. These results demonstrate that MALT1 protease activity contributes to T-dependent and T-independent antibody responses in vivo.

Case 2) Loss of binding ability (SH2-mediated): Human disease model for SHORT syndrome

Winnay JN, et al. PI3-kinase mutation linked to insulin and growth factor resistance in vivo. J Clin Invest. 2016.

SHORT syndrome is a disorder characterized by short stature, partial lipodystrophy, and insulin resistance.

The heterozygous mutation in the gene encoding the p85α regulatory subunit of PI3K, PI3KR1Arg649Trphas been identified in patients. This mutation occurs in the region encoding the C-terminal SH2 domain of p85α that is essential to bind PI3K to tyrosine phosphorylated proteins, and markedly attenuates the insulin-dependent activation of the PI3K pathway.

Model: Mice that are heterozygous for the p85R649W mutation, which is homologous to the mutation found in the majority of humans affected by SHORT syndrome.

Aim: Evaluate whether SHORT syndrome-associated PI3KR1 mutations account for the pathophysiology that underlies the abnormalities observed in SHORT syndrome patients.

Results: Similar to the patients, mutant mice exhibited a reduction in body weight and length, partial lipodystrophy, and systemic insulin resistance.

Figure 1. Decreased body weight and length in p85WT/R649W mice.

Figure 1 - p85R649W mice

A) Representative images of female p85αWT/WT (left) and p85αWT/R649W (right) mice at 24 weeks of age.

B) Body weight of p85αWT/WT (white) and p85αWT/R649W (red) mice measured over the indicated time course. Female mice are indicated by triangular symbols, and male mice are indicated by circular symbols.

C-E) Body weight at 24 weeks of age, and body length, and tibia length of 12-week-old male animals.

Figure 2. Heterozygous mutant mice are insulin resistant.

Figure 2 - p85R649W mice

A) Fed p85αWT/R649W mice exhibited marked hyperglycemia when compared with wt mice.

B) Fed serum insulin levels were markedly elevated in p85αWT/R649W mice.

C) Increase in insulin resistance, as measured by the homeostasis model assessment of insulin resistance (HOMA-IR), which was significantly increased in mutant mice.

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