During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here, we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin ؉ lymph sacs and cardinal veins, but not in podoplanin ؊/؊ embryos. Thus, podoplanin ؊/؊ mice develop a "nonseparation" phenotype, characterized by a bloodfilled lymphatic network after approximately embryonic day 13.5, which, however, partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplaninblocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system. (Blood. 2010;115(19):3997-4005)

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Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

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