The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. Using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or mRNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25-100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within four months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.The laboratory rat is a well-established model for the genetic dissection of human diseaserelated traits (1) despite the fact that targeted modification of its genome is largely intractable. We investigated the application of engineered zinc-finger nucleases (ZFNs;(2)) for the elimination of specific rat gene function and generation of "knockout" rats. ZFNs induce sitespecific, double-strand DNA breaks that can be repaired by the error-prone non-homologous end joining DNA repair pathway to result in a targeted mutation (Fig. 1A). In the fruit fly and zebrafish, direct embryo injection of ZFN-encoding mRNA has been used to generate heritable knockout mutations at specific loci (2).The design and validation of ZFN reagents to target a single-copy Green Fluorescent Protein (GFP) transgene inserted in a rat chromosome and two endogenous rat genes, IgM and

Disruption of the mouse gene encoding the blood coagulation inhibitor thrombomodulin (Thbd) leads to embryonic lethality caused by an unknown defect in the placenta. We show that the abortion of thrombomodulin-deficient embryos is caused by tissue factor-initiated activation of the blood coagulation cascade at the feto-maternal interface. Activated coagulation factors induce cell death and growth inhibition of placental trophoblast cells by two distinct mechanisms. The death of giant trophoblast cells is caused by conversion of the thrombin substrate fibrinogen to fibrin and subsequent formation of fibrin degradation products. In contrast, the growth arrest of trophoblast cells is not mediated by fibrin, but is a likely result of engagement of protease-activated receptors (PAR)-2 and PAR-4 by coagulation factors. These findings show a new function for the thrombomodulin-protein C system in controlling the growth and survival of trophoblast cells in the placenta. This function is essential for the maintenance of pregnancy.

IntroductionIn the hemochorial type of placentation observed in humans and mice, fetal nutrition involves the direct uptake of nutrients by zygote-derived trophoblast cells from circulating maternal blood. The required placental morphology is achieved through a highly regulated process of trophoblast differentiation coupled with remodeling of maternal and fetal vasculature. As a consequence, in contrast to all other vascular beds in which the blood vessel endothelium is the principal gatekeeper between tissue and blood, the terminal vascular space of the placenta is lined by trophoblast cells. 1,2 Trophoblast cells are genetically distinct from the maternal vascular endothelium and are derived from a different developmental lineage than endothelial cells. 3 In all nonplacental vascular beds, normal endothelium proactively suppresses the activity of the coagulation system, thereby maintaining a nonthrombogenic surface. A survey of existing data suggests that trophoblast cells also produce endothelial regulators of hemostasis, such as thrombomodulin (TM), endothelial protein C receptor (EPCR), and tissue factor pathway inhibitor (TFPI). [4][5][6][7][8] Such findings indicate that trophoblast cells might exhibit an endothelial cell-like ability to partake in the regulation of hemostasis at the fetomaternal interface. Indeed, the term "endothelial mimicry" has been coined to describe a process of remodeling of the maternal arteries, during which so-called "endovascular" trophoblast cells replace the maternal endothelium in these blood vessels and switch their expression from epithelial to endothelial adhesion receptor repertoire. [9][10][11] It is unknown whether trophoblast cells acquire anticoagulant gene expression in a temporally and spatially controlled manner similar to that described for the subset of endovascular trophoblast cells or whether the acquisition of an endothelial cell-like anticoagulant phenotype is a cell type-defining feature of trophoblast cells in general.The placenta also is a rich source of the initiator of coagulation, tissue factor (TF). TF antigen and procoagulant activity are detected in mouse giant and labrynthine trophoblasts and on human syncytiotrophoblast membranes. [12][13][14][15] With the exception of angiogenic endothelium, and in endothelium subjected to inflammatory and thrombotic stimuli, TF expression is strictly excluded from endothelial cells. Proinflammatory cytokines, ligands for Tollreceptors, and the principal coagulation protease, thrombin, induce TF expression in cultured endothelial cells, evoke increased production of endothelial-leukocyte adhesion receptors, and simultaneously suppress the expression of anticoagulant gene products. This transition from a noncoagulant and antiadhesive phenotype to a state of enhanced coagulation and leukocyte interactions has been termed "endothelial activation" and appears to reflect a principal switch in a concerted gene-expression program. 16 In contrast, trophoblast cells constitutively express TF, thus exhibiting, even under ...

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd−/−) mice to term. Half of the Thbd−/− offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd−/− animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd−/− mice. However, Thbd−/− females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy.