Constitutive Knockout Mouse | To get faster first results

A constitutive Knockout mouse, also referred to as a conventional or whole-body Knockout (KO), defines a mouse model in which the target gene is permanently inactivated in the whole animal, in every cell of the organism.

This gene inactivation is achieved at all stages of development, from the one-cell embryo stage through adulthood.

Applications

For academic research:

Determine main functions of the gene and/or protein
Study human pathologies caused by gene inactivation or deficiency
Induce a phenotype to create a disease model or in vivoresearch tool

For bio-pharmaceutical research & development:

Validate target gene
Study specificity and/or off-target activities of drug candidate
Develop new antibodies on the target protein
Safety and toxicology studies

Strengths of constitutive Knockout mouse models

Total absence of protein including all isoforms, or total absence of specific isoforms
Fast solution for preliminary in vivo studies
Feasible in all genetic backgrounds

Limitations of constitutive Knockout mouse models

Causes embryonic lethality in about 15% of all cases
May modify the animal physiology, adaptation and compensation mechanisms, resulting in false results
Global phenotype is the combination of different constitutions in different tissues that may lead to non-conclusive studies

→ These limitations can be bypassed by applying conditions such as time- or tissue-specificity

Case study

Model for idiopathic Parkinson’s disease (idPD)

‍Zhou Q, Yen A, Rymarczyk G, Asai H, Trengrove C, Aziz N, Kirber MT, Mostoslavsky G, Ikezu T, Wolozin B, Bolotina VM.
Impairment of PARK14-dependent Ca(2+) signalling is a novel determinant of Parkinson's disease.
Nat Commun. 2016.

Cells from idPD patients reveal a significant deficiency in store-operated PLA2G6-dependent Ca2+ signaling. The Pla2g6 gene (PARK14 disease locus) is poorly understood.

Model: Mice that constitutively lack the Pla2g6 gene mimicking the pathology observed in idPD patients.

Aim: Explore the origins of human age-dependent PD from the new perspective of PARK14 and the store-operated Ca2+ signaling, opening new opportunities for finding a cure for idPD.

Results: The molecular impairment of PLA2G6-dependent Ca2+ signaling triggers a sequence of pathological events of autophagic dysfunction, progressive loss of dopaminergic (DA) neurons in substantia nigra pars compacta and age-dependent L-DOPA-sensitive motor dysfunction.

Figure 1. PLA2G6 KO mice mimic store-operated Ca2+ entry (SOCE) deficiency in idPD patients.

A) Live primary skin fibroblasts from idPD and control donors were pretreated with thapsigargin (TG) to induce Ca2+ influx prior to Ca2+ addition. On average, there was more than 40% reduction in TG-induced Ca2+ influx in idPD patients compared with the control group.

‍B-C) MEFs (B) and iPSC-derived DA neurons (C) from WT and PLA2G6 KO mice were pretreated with TG prior to CA2+ addition. Impairment of TG-induced Ca2+ influx in the PLA2G6 KO mice mimics the defect found in fibroblasts of idPD patients.

Figure 2. PLA2G6 KO mice develop age-dependent PD-like phenotype.

Progressive motor dysfunction in aging KO, but not in WT mice developed at an age range that aligns with that typical of idPD in humans. Human age equivalent is shown below.