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

谷胱甘肽过氧化物酶（gpx）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。哺乳动物细胞中至少有四种不同的gpx和两种不同的trxr，它们在表达位点、细胞内定位和/或底物特异性方面存在差异。gpx以减少谷胱甘肽（GSH）为代价来减少过氧化物。氧化谷胱甘肽（GSSG）通过谷胱甘肽还原酶和NADPH循环为谷胱甘肽。磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPx）在谷胱甘肽过氧化物酶中是独一无二的，因为它具有底物特异性，定位于包括线粒体在内的各种位点，并且在硒缺乏时在硒蛋白的层次结构中处于较高的位置。PHGPx是唯一能够还原磷脂氢过氧化物的谷胱甘肽过氧化物酶，也接受低密度脂蛋白氢过氧化物和胆固醇氢过氧化物作为底物。硫氧还蛋白还原酶，通过氧化NADPH，再生硫氧还蛋白作为电子供体在各种氧化还原反应。已知的两种硫氧还蛋白还原酶（TRxR2）之一定位于线粒体。两种硒蛋白PHGPx和TRxR2与线粒体过氧化物还氧蛋白和超氧化物歧化酶一起，代表了线粒体抵御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。为了研究PHGPx和TRxR2在细胞水平上的表达模式并阐明其在体内的功能，我们提出(i)将lacZ基因插入到基因组中的PHGPx和TRxR2位点，（ii）在小鼠中敲除这两个基因。由于PHGPx和TRxR2可能是至关重要的，我们还将（iii）生成条件PHGPx和TRxR2等位基因，并尝试使用CRE-loxP系统以组织特异性的方式敲除小鼠体内的基因。我们将研究PHGPx和TRxR2 k.o.小鼠对氧化应激的敏感性和诱导细胞凋亡。

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.