Direct Comparison of a Natural Loss-Of-Function Single Nucleotide Polymorphism with a Targeted Deletion in the Ncf1 Gene Reveals Different Phenotypes

Abstract

The genetic targeting of mouse models has given insight into complex processes. However, phenotypes of genetically targeted mice are susceptible to artifacts due to gene manipulation, which may lead to misinterpretation of the observations. To directly address these issues, we have compared the immunological phenotypes of Ncf1 knockout mice with Ncf1 m1J mice possessing a naturally occurring intronic loss-of-function SNP in their Ncf1 gene. Neutrophil cytosolic factor 1 (NCF1) is the key regulatory component of the phagocytic NADPH oxidase 2 (NOX2) complex. Defects in NCF1 lead to lower production of reactive oxygen species (ROS) associated with autoimmune diseases in humans. In mice, collagen induced arthritis (CIA) and psoriatic arthritis are autoimmune disorders known to be regulated by Ncf1, and they were utilized in the present study to compare the Ncf1 knockout with Ncf1 m1J mice. Targeted Ncf1 knockout mice were generated on a pure C57BL/6N genetic background, and thereafter crossed with B10.Q.Ncf1 m1J mice. The targeting silenced the Ncf1 gene as intended, and both the B6N;B10.Q.Ncf1 m1J mice as well as the knockout littermates had reduced ROS production compared to wild type mice. Both also exhibited enhanced STAT1 (signal transducer and activator of transcription 1) protein expression as an indicator of pronounced interferon signature reported recently for Ncf1 deficient mice. Surprisingly, female Ncf1 knockout mice were protected from CIA whereas the Ncf1 m1J females developed severe disease. Ovariectomization retrieved the susceptibility of Ncf1 knockout females pointing to a sex hormone regulated protection against CIA in these mice. The data partly explains the discrepancy of the phenotypes reported earlier utilizing the Ncf1 m1J mice or Ncf1 knockout mice. These observations indicate that even a targeted knockout mutation may lead to a different biological outcome in comparison to the natural loss-of-function mutation of the same gene.

Chronic Active Arthritis Driven by Macrophages Without Involvement of T Cells

“…All mice were housed in specific pathogen–free conditions in either open cages or closed cages , as previously described, and were provided with enrichments, standard chow, and water ad libitum at the Central Animal Laboratory of the University of Turku or the Medical Inflammation Research animal house at the Karolinska Institute. All experiments were carried out with ethics approval from the University of Turku (permit nos.…”

Rheumatoid arthritis: identifying and characterising polymorphisms using rat models

“…For example, Ncf1 knockout mice were reported to be completely protected from EAE (van der Veen et al, 2000), whereas naturally occurring Ncf1 loss-of-function mutant mice developed enhanced EAE (Hultqvist et al, 2004). Female Ncf1 knockout mice were protected from CIA, whereas female Ncf1 mutant mice developed severe CIA (Sareila et al, 2015). These phenotypic differences can be due to the targeted gene being closely flanked by regions of donor origin in genetically modified mice despite repeated backcrossing to the host strain (Vanden Berghe et al, 2015).…”

The Macrophage Mannose Receptor Regulate Mannan-Induced Psoriasis, Psoriatic Arthritis, and Rheumatoid Arthritis-Like Disease Models

“…If not otherwise noted, littermates were used in the experiments. Mice were housed under specific pathogen-free conditions as described earlier ( 25 ). The study was approved by the National Animal Experiment Board in Finland, ethical permit numbers ESAVI-0000497/041003/2011 and ESAVI/439/04.10.07/2017.…”