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

在过去的几年中，我们已经克隆，表达克隆，磷脂氢过氧化物谷胱甘肽过氧化物酶基因（PHGPx）作为一个基因，保护伯基特淋巴瘤细胞凋亡。PHGPx是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关键作用，因此敲除（k.o.）PHGPx基因在小鼠中的研究已经开始。因为没有办法预测PHGPx基因对存活和生育力的重要性，也没有办法预测酶促氧化还原系统的冗余，所以平行地遵循几种策略，包括直接k.o.和条件k.o.这两种方法都没有引起靶向PHGPx等位基因的生殖系传播。又一次尝试生成条件k.o.当Behne（柏林）的小组和我们的小组发现了携带在第一次尝试产生条件性k.o.这种新形式的PHGPx编码一种新的精子特异性核形式的PHGPx，其很可能在精子核的染色质凝聚中起关键作用。虽然PHGPx k.o.在进行中，Ursini和Flohé的小组发现了PHGPx的另一种功能，并确定PHGPx是精子线粒体囊的主要成分。第二个条件k.o.该策略成功地产生了在生殖系中具有loxP侧翼（floxed）PHGPx等位基因的小鼠。在新的资助期内，我们的目标首先是从分子上表征新PHGPx的信使RNA，包括转录起始位点。我们的第二个主要目的是产生和表征在不同组织中缺乏功能性PHGPx基因的小鼠。作为第一步，应通过将具有floxed PHGPx等位基因的小鼠与普遍表达Cre重组酶的“删除”小鼠杂交来产生PHGPx半合子小鼠，并应研究半合子小鼠的表型。特别感兴趣的是针对半合子是否与男性不育和睾丸萎缩的问题。作为第二步，我们将以组织特异性方式删除第二个PHGPx基因。特别强调将致力于PHGPx无效表型在脑，内皮和睾丸的分析。通过将具有floxed 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以及它起什么作用的问题。

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.