A point mutation Knockin mouse defines an animal model in which one or more nucleotides are constitutively mutated.

The insertion, deletion, nonsense and sense mutations can alter the amino acid sequence of a given protein, and so dramatically affect its function.


For academic research:

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

For bio-pharmaceutical research & development:

  • Study drug-resistant mutants
  • Alter drug-antibody affinities
  • Pharmacological off-target and efficacy studies
  • Mimic human genetic diseases

Strengths of point mutation Knockin 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 KO where the whole gene is deleted
  • Phenotype due only to the mutation: alteration of a single function without disturbing other domains of a protein

Limitations of point mutation Knockin 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 gene inactivation
  • 1. Modification or disruption of splicing regulation
    2. Genetic redundancy
    →  Can be assessed via constitutive Knockout of the gene of interest

Case Study

Model with heterozygous point mutations, exactly homologous to human retinitis pigmentosa (RP).

Sothilingam V, et al. Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype. Hum Mol Genet. 2015.

Mutations in the PDE6A gene can cause rod photoreceptor degeneration and the blinding disease retinitis pigmentosa.

Model: Novel mouse model for the Pde6aR562W point mutation in combination with an existing line carrying the V685Mpoint mutation to generate compound heterozygous Pde6aV685M/R562W animals, exactly homologous to a case of human RP.

Aim: Predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window of opportunity for clinical interventions.

Results: The study provides a rational basis for predictions on human RP phenotypes and disease progression in compound heterozygous situations, and suggests the targeting of non-apoptotic processes as a feasible treatment approach.

Figure 1. Loss of PDE6A expression causes cGMP accumulation.

Excessive accumulation of cGMP and subsequent rod photoreceptor death, followed by a mutation-independent, secondary death of cone photoreceptors.

Figure 1a-d - Pde6aR562W mice

A, B) In the PN11 wildtype (wt) retina (A), immunostaining for PDE6A shows strong protein expression in photoreceptor OS.

In contrast, at the same age, in the compound heterozygous V685M/R562W retina (C), the protein is essentially absent.

C, D) At PN11, wt retina is essentially negative for cGMP immunoreactivity (C).

PDE6A mutants, however, display individual rod photoreceptor cells that have accumulated large amounts of cGMP (D).

Figure 1e - Pde6aR562W mice

E) The quantification of cGMP-positive cells in the ONL and the PDE6A pixel intensity in the OS (arbitrary units; AU) shows an inverse correlation. Images are representative for immunostaining performed on retinal sections from at least three independent animals for each genotype.

Figure 2. Photoreceptor cell death and survival.

Figure 2a-f - Pde6aR562W mice

A-C) The TUNEL assay in the wt retina occasionally labeled cells dying due to developmental processes.

D-F) In Pde6a mutants, photoreceptor cell death was dramatically increased.

The images show the situation at P12, P15 and P21, time-points corresponding to the peak of cell death..

G, H) The line graph at the bottom left (G) illustrates the progression of photoreceptor cell death as evidenced by the TUNEL assay in different Pde6a mutants. The green line corresponds to the V685M*R562W mutant.

The peak times as well as the peak amplitudes correspond to the speed of retinal degeneration, which is illustrated by the loss of photoreceptors (H).

Images are representative for TUNEL assays performed on retinal sections from at least three independent animals; quantifications in G, H include data from 3-7 animals per genotype and time-point.

Figure 2gh - Pde6aR562W mice

Figure 3. Photoreceptor degeneration in Pde6a mutants correlates with calpain, not caspase, activity.

Calpain activity is often associated with non-apoptotic forms of cell death, and in all Pde6a mutants it is strongly correlated in time with the progression of photoreceptor cell death.

Figure 3ab - Pde6aR562W mice

A) Quantification of caspase-3-positive cells, which shows only extremely low numbers of cells at the respective peaks of degeneration, with no significant (n.s.) differences to wt.

B) The progression of calpain activity was analyzed over time and showed a strong correlation to the extent of cell death and the progression of retinal degeneration.

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