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

在过去的几年里，我们通过表达克隆的方法克隆了磷脂过氧化氢谷胱甘肽过氧化物酶基因(PHGPx)，它是一种保护Burkitt淋巴瘤细胞免受凋亡的基因。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节中起着关键作用，因此基因敲除(k.o.)PHGPx基因在小鼠体内的研究已经开始。由于无法预测PHGPx基因对生存和生育的重要性，也无法预测酶氧化还原系统的冗余性，因此采取了几种并行的策略，包括直接的k.o。和有条件的K.O。这两种方法都没有引起目标PHGPx等位基因的胚系传播。又一次试图产生有条件的k.o。当Behne(柏林)和我们的团队发现一种新形式的PHGPx携带替代外显子时，使用修改的策略变得必要，该外显子在第一次尝试产生条件k.o时被摧毁。这种新形式的PHGPx编码了一种新的精子特异性核形式PHGPx，它很可能在精子核的染色质凝聚中发挥关键作用。而PHGPx k.O.在此过程中，Ursini和Flohé小组发现了PHGPx的另一种功能，并确定PHGPx是精子线粒体被膜的主要成分。第二个有条件的K.O.策略是成功地在生殖系中产生了带有loxP侧翼(花环)PHGPx等位基因的小鼠。在新的资助期，我们首先致力于从分子上表征新的PHGPx的信使RNA，包括转录起始点。我们的第二个也是主要的目标是培育和鉴定在不同组织中缺乏功能性PHGPx基因的小鼠。作为第一步，PHGPx基因的半合子小鼠将通过将带有PHGPx等位基因的小鼠与无处不在表达Cre重组酶的小鼠杂交而产生，并对半合子小鼠的表型进行研究。人们特别感兴趣的问题是，偏侧是否与男性不育和睾丸萎缩有关。作为第二步，我们将以组织特异性的方式删除第二个PHGPx基因。将特别强调PHGPx缺失表型在脑、内皮和睾丸中的分析。通过将携带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，问题将被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.