Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies

The use of nuclear transfer to produce transgenic pigs

Manipulation of the pig genome has the potential to improve pig production and offers powerful biomedical applications. Genetic manipulation of mammals has been possible for over two decades, but the technology available has proven both difficult and inefficient. The development of new techniques to enhance efficiency and overcome the complications of random insertion is of importance. Nuclear transfer combined with homologous recombination provides a possible solution: precise genetic modifications in the pig genome may be induced via homologous recombination, and viable offspring can be produced by nuclear transfer using cultured transfected cell lines. The technique is still ineffective, but it is believed to have immense potential. One area that would benefit from the technology is that of xenotransplantation: transgenic pigs are expected to be available as organ donors in the foreseeable future

Capacitative calcium entry mechanism in porcine oocytes

The presence of the capacitative Ca2+ entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca2+-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca2+ entry. A similar divalent cation influx could also be detected with the Mn2+-quench technique after inositol 1,4,5-triphosphate-induced Ca2+ release. In both cases, lanthanum, the Ca2+ permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca2+ influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca2+ entry mechanism might help in refilling the intracellular stores after the release of Ca2+ from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca2+ entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca2+ entry mechanism and thus contributes to Ca2+ influx

Na\u3csup\u3e+\u3c/sup\u3e/Ca\u3csup\u3e2+\u3c/sup\u3e exchanger in porcine oocytes

The presence of the Na+/Ca2+ exchange mechanism was investigated in porcine oocytes. Immature and in vitro-matured oocytes were loaded with the Ca2+-sensitive fluorescent dye fura 2 and changes in the intracellular free Ca2+ concentration ([Ca2+]i) were monitored after altering the Na+ concentration gradient across the plasma membrane. Decreasing the extracellular Na+ concentration induced an increase in [Ca2+]i possibly by a Ca2+ influx via the Na+/Ca2+ exchanger. A similar Ca2+ influx could also be triggered after increasing the intracellular Na+ concentration by incubation in the presence of ouabain (0.4 mM), a Na+/K+-ATPase inhibitor. The increase in the [Ca2+]i was due to Ca2+ influx since it was abolished in the absence of extracellular Ca2+, and the increase was mediated by the Na+/Ca2+ exchanger since it was blocked by the application of amiloride or bepridil, inhibitors of Na+/Ca2+ exchange. Verapamil (50 μM) and pimozide (50 μM), inhibitors of L- and T-type voltage-gated Ca2+ channels, respectively, could not block the Ca2+ influx. The Ca2+ entry via the Na+/Ca2+ exchanger could not induce the release of cortical granules and did not stimulate the resumption of meiosis. This was unexpected because Ca2+ is thought to be a universal trigger for activation. Using antibodies raised against the exchanger, it was demonstrated that the Na+/Ca2+ exchanger was localized predominantly in the plasma membrane. Reverse transcription-polymerase chain reaction revealed that porcine oocytes contain a transcript that shows 98.1% homology to the NACA3 isoform of the porcine Na+/Ca2+ exchanger