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NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein
Monika Göllner1, Irmgard Ihrig-Biedert1, Victoria Petermann1
1Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany.
Background:
The superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2 or gp91phox, the phagocytic isoform) was reported as a major source of oxidative stress in various human diseases. Genetic deletion is widely used to study the impact of NOX2-derived reactive oxygen species (ROS) on disease development and progression in various animal models. Here, we investigate why NOX2 knockout mice show no NOX2 activity but express NOX2 mRNA and protein.
Methods And Results:
Oxidative burst (NOX2-dependent formation of ROS) was measured by L-012-based chemiluminescence and was largely absent in whole blood of NOX2 knockout mice. Protein expression was still detectable in different tissues of the NOX2 knockout mice, at the expected and a slightly lower molecular weight (determined by Western blot). The NOX2 gene was even largely enhanced at its expressional level in NOX2 knockout mice. RNA sequencing revealed a modified NOX2 mRNA in the knockout mice that is obviously translated to a truncated inactive mutant enzyme.
Conclusion:
Although the commercial NOX2 knockout mice display no considerable enzymatic NOX2 activity, expression of the NOX2 gene (when using standard primers) and protein (when using antibodies binding to the carboxy-terminal end) can still be detected, which may lead to confusion among investigators.
Insights
NOX2 knockout mice lack enzymatic activity but still express NOX2 mRNA and protein due to a modified mRNA leading to a truncated enzyme. This can cause confusion in research on oxidative stress and disease models.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) is a key enzyme generating reactive oxygen species (ROS) and contributing to oxidative stress in diseases.
- Genetic knockout models are crucial for studying NOX2's role in disease pathogenesis.
- Investigating discrepancies in NOX2 knockout mice is essential for accurate research.
Purpose of the Study:
- To elucidate the reasons behind the lack of NOX2 enzymatic activity in NOX2 knockout mice despite the presence of NOX2 mRNA and protein.
- To understand the molecular mechanisms underlying the observed phenotype in NOX2 knockout models.
Main Methods:
- Measurement of oxidative burst using L-012-based chemiluminescence.
- Western blot analysis to detect NOX2 protein expression.
- RNA sequencing to analyze NOX2 gene and mRNA expression.
Main Results:
- NOX2 knockout mice exhibited minimal to no NOX2-dependent ROS production (oxidative burst).
- Western blot confirmed the presence of NOX2 protein in knockout mice, albeit at a slightly altered molecular weight.
- RNA sequencing identified a modified NOX2 mRNA transcript, leading to the translation of a truncated, inactive NOX2 enzyme.
- Gene expression analysis indicated an enhanced expression level of the NOX2 gene in knockout mice.
Conclusions:
- Commercial NOX2 knockout mice demonstrate negligible enzymatic NOX2 activity.
- Detection of NOX2 gene and protein in these models can occur due to standard detection methods, potentially misleading researchers.
- Understanding these molecular alterations is critical for the correct interpretation of studies using NOX2 knockout mice.
