Expression of biologically active human TRAIL in transgenic pigs.

BACKGROUND: Xenotransplantation of porcine organs into human recipients is a potential option for overcoming the dramatic shortage of suitable donor organs. To date, transgenic modification of pig organs has achieved partial or temporal reduction of xenograft rejection by inhibition of hyperacute rejection. Expression of human tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) in transgenic pigs might be a strategy for controlling posthyperacute rejection mechanisms mediated by cellular components of the immune system. The objective of this study was generation of a transgenic pig model to evaluate the potential of this strategy for xenotransplantation. METHODS: Transgenic pigs were generated by microinjection of an expression vector for human TRAIL under control of the murine H-2K promoter. Expression of the transgene was analyzed by Western blot and immunohistochemistry. Biologic activity of TRAIL on transgenic porcine lymphocytes was evaluated in co-culture experiments using Jurkat and Hut 78.2 cells as targets. RESULTS: In three lines of transgenic pigs, human TRAIL protein was detected in the membrane fractions of various tissues. Highest expression levels were observed in spleen and lung. Human TRAIL expression on porcine lymphocytes was augmented on activation of cells. Transgenic pig lymphoblasts induced apoptosis in Jurkat and Hut 78.2 cells, which was inhibited by neutralizing anti-TRAIL antibodies, demonstrating a TRAIL-specific effect. CONCLUSIONS: Ubiquitous expression of human TRAIL was achieved in transgenic pigs without detrimental side effects. Pigs expressing biologically active human TRAIL will be used for future xenotransplantation experiments to modulate primate anti-pig cellular immune responses.

Quantitative monitoring of pluripotency gene activation after somatic cloning in cattle.

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

Evaluation of laser-assisted lentiviral transgenesis in bovine.

Lentiviral transduction of oocytes or early embryos is an efficient strategy to generate transgenic rodents and livestock. We evaluated laser-based microdrilling (MD) of the zona pellucida, which is a physical barrier for viral infection, and subsequent incubation in virus suspension as a new route for lentiviral transgenesis in bovine. Lentiviral vectors carrying an eGFP expression cassette were used to transduce oocytes or zygotes after MD as compared to the established subzonal virus injection technique (MI). The type of manipulation (MD vs. MI) did not affect cleavage rates, but had a significant effect on blastocyst rates (P < 0.001). MI of virus or sham-MI (buffer) resulted in higher blastocyst rates as compared to MD, both in the oocyte and zygote treatment groups. The latter exhibited higher rates of early cleavage (P < 0.05) and blastocyst rates (P < 0.01). The proportion of eGFP expressing blastocysts was higher after infection of oocytes (MD: 44 +/- 9%; MI: 67 +/- 8%) than after infection of zygotes (MD: 26 +/- 8%; MI: 26 +/- 9%). Overall efficacy (eGFP-positive blastocysts per treated oocytes or zygotes) was highest after MI of oocytes (18 +/- 2%). Our study demonstrates the feasibility of laser-assisted lentiviral gene transfer into bovine oocytes and zygotes. However, further optimization of the procedure is required, mainly to reduce the incidence of polyspermy after MD of oocytes and to eliminate negative effects of MD on early embryonic development.