Göllner Monika, Ihrig-Biedert Irmgard, Petermann Victoria, Saurin Sabrina, Oelze Matthias, Kröller-Schön Swenja, Vujacic-Mirski Ksenija, Kuntic Marin, Pautz Andrea, Daiber Andreas, Kleinert Hartmut
Department of Pharmacology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany.
Laboratory of Molecular Cardiology, Department of Cardiology 1, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany.
Antioxidants (Basel). 2020 Oct 26;9(11):1043. doi: 10.3390/antiox9111043.
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.
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.
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.
超氧化物生成酶烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶（NOX2或gp91phox，吞噬异构体）被报道为多种人类疾病中氧化应激的主要来源。基因敲除被广泛用于研究NOX2衍生的活性氧（ROS）对各种动物模型中疾病发展和进程的影响。在此，我们研究了为什么NOX2基因敲除小鼠没有NOX2活性，但却表达NOX2 mRNA和蛋白质。
通过基于L-012的化学发光法测量氧化爆发（依赖NOX2的ROS形成），NOX2基因敲除小鼠的全血中基本不存在这种现象。在NOX2基因敲除小鼠的不同组织中仍可检测到蛋白质表达，其分子量与预期相符且略低（通过蛋白质印迹法测定）。在NOX2基因敲除小鼠中，NOX2基因的表达水平甚至大幅提高。RNA测序揭示了敲除小鼠中一种修饰的NOX2 mRNA，它明显被翻译为一种截短的无活性突变酶。
尽管市售的NOX2基因敲除小鼠没有可观的NOX2酶活性，但仍可检测到NOX2基因的表达（使用标准引物时）和蛋白质（使用与羧基末端结合的抗体时），这可能会给研究人员带来困惑。
