Kenneth R. Bondioli

Production of α1,3-Galactosyltransferase-Knockout Cloned Pigs Expressing Human α1,2-Fucosylosyltransferase

Abstract 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 α1,3-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 α1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an α1,3-GT knockout allele and express a randomly inserted human α1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous α1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the α1,3-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.

Expression of the human α1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis

Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell‐based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human α1,2‐fucosyltransferase (H‐transferase, HT). H‐transferase expression modifies the cell surface carbohydrate phenotype of the xe‐nogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Galα1,3‐Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine‐inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Galα1,3‐Gal epitope, reduced XNA reactivity, and an increased resistance to human serum‐mediated cytolysis. Transgenic pigs that express H‐transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.—Costa, C., Zhao, L., Burton, W. V., Bondioli, K. R., Williams, B. L., Hoagland, T. A., DiTullio, P. A., Ebert, K. M., Fodor, W. L. Expression of the human α1,2‐fucosyltrans‐ferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum‐mediated cytolysis. FASEB J. 13, 1762–1773 (1999)

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection

Abstract: Research in pig‐to‐primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell‐ and tissue‐based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human α1,2‐fucosyltransferase (H transferase, HT) in pigs modifies the cell‐surface carbohydrate phenotype resulting in reduced Galα1,3‐Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum‐mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro‐inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.

Capacitative Calcium Entry Mechanism in Porcine Oocytes

The presence of the capacitative Ca21 entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca21-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 Ca21 entry. A similar divalent cation influx could also be detected with the Mn21quench technique after inositol 1,4,5-triphosphate-induced Ca21 release. In both cases, lanthanum, the Ca21 permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca21 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 Ca21 entry mechanism might help in refilling the intracellular stores after the release of Ca21 from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca21 entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca21 entry mechanism and thus contributes to Ca21 influx. calcium, embryo, fertilization, ovum

Na+/Ca2+ Exchanger in Porcine Oocytes

Abstract 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.

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.

Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had > or = 8 cells at 84 hpa, while 32% of the bovine NT embryos had > or = 8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had > or = 8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further.