Project Details
Projekt
Characterization of mice lacking functional phospholipid hydroperoxide glutathione peroxidase (PHGPx): tissue specific gene targeting
Applicant
Professor Dr. Georg Wilhelm Bornkamm
Subject Area
Biochemistry
Term
from 2002 to 2008
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 5366945
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.
DFG Programme
Priority Programmes
Participating Person
Dr. Marcus Conrad
