NOX2ko Mice Show Largely Increased Expression of a Mutated NOX2 mRNA Encoding an Inactive NOX2 Protein.

Abstract

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.