Targeted disruption of the mouse protein C inhibitor gene and its consequences on the haemostatic system, kallilrein-kinin system and reproduction system

Protein C inhibitor (PC) is a serine protease inhibitor that inhibits a variety of plasmatic and extravascular serine proteases by forming enzymatically inactive stable 1:1 complexes. Serine proteases inhibited by PC include the anticoagulatory enzyme activated protein C, clotting factors Xa, XIa, plasma kailikrein and thrombin, the plasminogen activators urokinase and tissue plasminogen activator, as well as the trypsin-like, extravascular proteases tissue kallikrein, prostate specific antigen and acrosin. So far, however, the physiological role of PCI has not been elucidated. It is therefore the aim of this proposal to study the effect of the knockout of PCI by using the transgenic approach. A targeting vector, pPNT.PCI consisting of 9.2 kb homologous sequence of mouse PCI gene, a positive selection marker (neomycine resistance gene) and a negative selection marker (thymidine kinase) gene was prepared. Electroporation of R1 embryonic stem (ES) cells with the linearized pPNT.PCI yielded five targeted clones with correct homologous recombination at the 5- and 3- ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones were used to generate PCI deficient mice. Two chimeric mice (one male and female), with chimerism ranging from 60-80 % were born and these mice were already mated to obtain heterozygots (PCI+/-) transgenes. The founders chimeric mice will be followed until they die to determine the lifespan relative to wild type. The f1 stock will be crossed to produce homozygous transgenes for analysis (PCI-/-). Homozygot mice will be tested for the presence of the targeted gene by Southern hybridization using the DNA from a tail clip. Adults of the transgenic strain(s) of interest will be assayed by Northern and Western blotting on tissue extracts to determine the level of PCI mRNA and protein. The lifespan, the body weight at 5 weeks of age and the frequency of their litters will be evaluated in wild-type and homozygous transgenic animals. The animals will be tested with respect to the tissue damage, hormone delivery, haemostatic system, kallikrein - kinin system and the reproduction system, as well. Because of the anticipated important role of PCI in the inhibition of the activities of serine proteases, successful solution of the proposed project will further elucidate the role of this protein.

Protein C inhibitor (PCI) is a serine protease inhibitor that inhibits a variety of plasmatic and extravascular serine proteases by forming enzymatically inactive stable 1:1 complexes. Serine proteases inhibited by PCI include the anticoagulatory enzyme activated protein C, clotting factors Xa, XIa, plasma kallikrein and thrombin, the plasminogen activators urokinase and tissue plasminogen activator, as well as the trypsin-like, extravascular proteases tissue kallikrein, prostate specific antigen and acrosin. So far, however, the physiological role of PCI has not been elucidated. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. Within this project we created and characterized transgenic mice with inactivated protein C inhibitor. Male PCI deficient mice were infertile, in spite of their normal sexual activity. Performed spermiograms revealed that more than 95% of sperm obtained from the epididymis of PCI-/- mice were morphologically abnormal; most lacked tails and were degenerated. Morphological analysis revealed the destruction of the Sertoli cells and of the blood-testis barrier in PCI deficient males, presumably as a consequence of unbalanced proteolytic activity, as revealed using chromogenic substrates. The above given results were published in the Journal of Clinical Investigation (current impact factor 12.015). This article was so far cited in 7 top-ranking journals (with average impact factor 4.77). Further we characterized the expression of PCI during the mouse development (manuscript in preparation). Lately in older mice we observed increased occurrence of spontaneously developed tumours while no tumors in the control wild-type mice were detected. Within the project we established efficient cooperation with the Center for Transgene Technology & Gene Therapy in Leuven, Belgium and we were able to build our gene-knockout facility in our institute of Vascular Biology and Thrombsosis Research. Our transgenic facility is the only one at the University of Vienna and it represents one of the very few such facilities that successfully accomplished gene targeting in mice in Austria. In summary, we believe that the outcome of our FWF project - where we could show that PCI is an absolute requirement for male fertility - is relevant and it creates a solid basis for analyzing more precisely the molecular bases of mouse and human infertility. Additionally, our preliminary results indicate possble role of PCI in tumor formation.