Charakterization of mice lacking functional mitochondrial thioredoxin reductase (TR2): tissue-specific gene targeting

谷胱甘肽过氧化物酶（GPxs）和硫氧还蛋白还原酶（TRxRs）是硒蛋白，在保护细胞免受氧化损伤中起着至关重要的作用。在哺乳动物细胞中存在至少四种不同的GPx和两种不同的TRxR，它们在表达位点、细胞内定位和/或底物特异性方面不同。GPx以还原型谷胱甘肽（GSH）为代价还原过氧化物。氧化型谷胱甘肽（GSSG）通过谷胱甘肽还原酶和NADPH再循环为GSH。磷脂氢谷胱甘肽过氧化物酶（PHGPx）是谷胱甘肽过氧化物酶中独特的一种，它具有底物特异性，在包括线粒体在内的多个位点都有定位，并且在缺硒时在硒蛋白的层次结构中占有较高的位置。PHGPx是唯一一种能够还原磷脂过氧化氢，并接受LDL过氧化氢和胆固醇过氧化氢作为底物的谷胱甘肽过氧化物酶。硫氧还蛋白还原酶通过氧化NADPH再生硫氧还蛋白，硫氧还蛋白在各种氧化还原反应中充当电子供体。两种已知的硫氧还蛋白还原酶之一（TRxR 2）定位于线粒体。两个硒蛋白PHGPx和TRxR 2，与线粒体过氧化物氧还蛋白和超氧化物歧化酶一起，代表线粒体中对抗由呼吸链作为氧还原副产物产生的活性氧中间体的主要防线。为了在细胞水平上研究PHGPx和TRxR 2的表达模式并阐明它们在体内的功能，我们建议（i）将lacZ基因插入到基因组PHGPx和TRxR 2位点中，以及（ii）在小鼠中敲除这些基因。由于PHGPx和TRxR 2可能是必需的，我们还将（iii）产生条件性PHGPx和TRxR 2等位基因，并将尝试使用CRE-loxP系统以组织特异性方式在小鼠体内敲除基因。我们将研究PHGPx和TRxR 2 k.o.小鼠氧化应激和诱导细胞凋亡。

Glutathione peroxidases (GPxs) and thioredoxin reductases (TRxRs) are selenoproteins playing a crucial role in protecting cells from oxidative damage. There are at least four different GPxs and two different TRxRs in mammalian cells which differ in their site of expression, intracellular localization and/or substrate specificity. GPxs reduce peroxides at the expense of reduced glutathione (GSH). Oxidized glutathione (GSSG) is recycled to GSH through glutathione reductase and NADPH. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is unique among the glutathione peroxidases for its substrate specificity, its localization at various sites including mitochondria and its high position in the hierarchy of selenoproteins during selenium deprivation. PHGPx is the only glutathione peroxidase that is able to reduce phospholipid hydroperoxides and that also accepts LDL hydroperoxides and cholesterol hydroperoxides as substrates.Thioredoxin reductases, by oxidizing NADPH, regenerate thioredoxins which serve as electron donors in a variety of redox reactions. One of the two known thioredoxin reductases (TRxR2) is localized to mitochondria. The two selenoproteins PHGPx and TRxR2, together with mitochondrial peroxiredoxin and superoxide dismutase, represent the primary line of defense in mitochondria against reactive oxygen intermediates produced by the respiratory chain as a side product of oxygen reduction. To study the expression pattern of PHGPx and TRxR2 at a cellular level and to elucidate their function in vivo, we propose to (i) insert the lacZ gene into the genomic PHGPx and TRxR2 loci and (ii) to knock out the genes in mice. Since PHGPx and TRxR2 might be vitally required, we shall also (iii) generate conditional PHGPx and TRxR2 alleles and shall attempt to knock out the genes in mice in vivo in a tissue specific manner using the CRE-loxP system. We shall investigate the susceptibility of PHGPx and TRxR2 k.o. mice to oxidative stress and the induction of apoptosis.