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

Production of α1,3-galactosyltransferase-knockout cloned pigs expressing human α1,2-fucosylosyltransferase

The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galα1,3-Gal epitope, which is synthesized by α1,3-galactosyltransferase (α1,3-GT). The Galα1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galα1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galα1,3-Gal antigen is the most attractive option. In this study, the 5′ end of the 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an 1,3-GT knockout allele and express a randomly inserted human 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection