These results are encouraging and warrant further studies on the biological function of heme oxygenase-I expression in hHO-1 transgenic pigs in the context of xenotransplantation.

The hA20 gene was for the first time functionally expressed in transgenic pigs. Although the CAGGS is a ubiquitous promoter element, expression was restricted to heart, skeletal muscle and PAECs of transgenic animals. Cultivated hA20-transgenic PAECs were protected against TNF-alpha-mediated apoptosis, and partially protected against CD95(Fas)L-mediated cell death; cardiomyocytes were partially protected in I/R. These findings reveal hA20 as a promising molecule for controlling AVR in multi-transgenic pigs for xenotransplantation studies.

For the first time, healthy hTM-transgenic pigs could be successfully generated by somatic cell nuclear transfer. hTM can be expressed in porcine organs without perturbation of the porcine coagulation system. hTM-transgenic porcine fibroblasts showed elevated aPC production in an in vitro hTM coactivity assay. These findings warrant further work on the control of the xenogenic activation of coagulation by transgenic approaches.

Injection of linearized DNA constructs into the pronuclei of fertilized mammalian eggs is a standard method for producing transgenic embryos and animals. Here, we show that injection of covalently closed circular (ccc) plasmids into the cytoplasm of fertilized bovine and murine eggs is a highly efficient and simple alternative for ectopic expression of foreign DNA in embryos. A broad range of plasmids could be successfully expressed in preimplantation stages, including plasmids and minicircles with a scaffold/matrix attachment region (S/MAR), conventional plasmids, and bacterial artificial chromosomes (BACs). Although the foreign DNA plasmids are mainly maintained as episomal entities during preimplantation development, they accurately behave like nuclear DNA. Onset of transcription of an Oct4 promoter-controlled marker gene coincided with the species-specific time points of major embryonic genome activation, and could be modulated by in vitro DNA-methylation. This approach allows an experimental access to reprogramming events in early mammalian embryos.

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.