Characterization of mice lacking functional phospholipid hydroperoxide glutathione peroxidase (PHGPx): tissue specific gene targeting

在过去的几年中，我们通过表达克隆克隆了磷脂氢过氧化物谷胱甘肽过氧化物酶基因（PHGPx），作为保护伯基特淋巴瘤细胞免于凋亡的基因。PHGPx是一种参与氧化还原代谢的硒酶。由于氧化还原代谢在细胞凋亡的调控中起着至关重要的作用，我们开始了小鼠PHGPx基因的敲除（k.o）方法。由于没有办法预测PHGPx基因对生存和生育的重要性，也没有办法预测酶促氧化还原系统的冗余性，因此同时采用了几种策略，包括直接k.o.和条件k.o.。这两种方法都没有引起目标PHGPx等位基因的种系传播。当behhne （Berlin）和我们的研究小组发现了一种新的PHGPx形式，这种形式的PHGPx携带一个在第一次产生条件k.o的尝试中被破坏的外显子时，有必要使用一种改进的策略来产生条件k.o。这种新的PHGPx形式编码了一种新的精子特异性核形式的PHGPx，这种形式很可能在精子核的染色质凝聚中起着至关重要的作用。在PHGPx项目进行中，Ursini和floh<e:1>的研究小组发现了PHGPx的另一种功能，并确定PHGPx是精子线粒体胶囊的主要成分。第二个条件k.o.策略在种系中成功地产生了具有loxP侧翼（floxed） PHGPx等位基因的小鼠。在新的资助期内，我们首先致力于表征新型PHGPx的信使RNA，包括转录起始位点。我们的第二个主要目标是在不同组织中产生和表征缺乏功能性PHGPx基因的小鼠。首先，将带固定PHGPx等位基因的小鼠与普遍表达Cre重组酶的小鼠杂交，产生PHGPx半合子小鼠，并对半合子小鼠的表型进行研究。特别感兴趣的是针对半合子性是否与男性不育和睾丸萎缩有关的问题。第二步，我们将以组织特异性的方式删除第二个PHGPx基因。重点分析PHGPx在脑、内皮和睾丸中的空表型。通过将带带PHGPx等位基因的小鼠与在精子发生的不同阶段表达Cre的小鼠杂交，将回答在精子发生的哪个阶段需要PHGPx以及它起什么作用。

During the last years we have cloned, by expression cloning, the phospholipid hydroperoxide glutathione peroxidase gene (PHGPx) as a gene that protects Burkitt's lymphoma cells from apoptosis. PHGPx is a selenoenzyme involved in redox metabolism. Since the redox metabolism plays a critical role in the regulation of apoptosis, a knock-out (k.o.) approach of the PHGPx gene in mice has been started. Because there was no way of anticipating the importance of the PHGPx gene for survival and fertility nor of predicting the redundancy of the enzymatic redox systems, several strategies were followed in parallel including a straight k.o. and a conditional k.o. Neither of both approaches gave rise to germ line transmission of the targeted PHGPx allele. Another attempt to generate a conditional k.o. using a modified strategy became necessary when the group of Behne (Berlin) and our group discovered a novel form of PHGPx carrying an alternative exon that had been destroyed in the first attempt to generate a conditional k.o. This novel form of PHGPx encodes a novel sperm-specific nuclear form of PHGPx that very likely plays a crucial role in chromatin condensation of sperm nuclei. While the PHGPx k.o. was in progress, the groups of Ursini and Flohé have found still another function of PHGPx and have identified PHGPx as a major constituent of the capsule of sperm mitochondria. The second conditional k.o. strategy was successful yielding mice with a loxP flanked (floxed) PHGPx allele in the germ line. In the novel funding period we are first aiming to characterize molecularly the messenger RNA of the novel PHGPx including the transcription start site. Our second and major aim is to generate and characterize mice lacking a functional PHGPx gene in different tissues. As a first step, mice hemizygous for PHGPx shall be generated by crossing mice with a floxed PHGPx allele to "deleter" mice expressing the Cre recombinase ubiquitously, and the phenotype of the hemizygous mice shall be studied. Particular interest is directed to the question whether hemizygosity is associated with male infertility and testicular atrophy. As a second step, we shall delete the second PHGPx gene in a tissue-specific manner. Special emphasis will be devoted to the analysis of the PHGPx null phenotype in brain, endothelium and testis. By crossing the mice with the floxed PHGPx allele with mice expressing Cre during different stages of spermatogenesis the question will be answered at which stage of spermatogenesis PHGPx is required and which function it serves.