Attempt at creating donor pig by nuclear transplantation for xenotrasplantation

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1) My colleges and I have attempted to create alpha-gal knockout pig. Now, we are not successful in it yet. 2) To examine the means to change the allo- or xeno-reactivity after transplantation, we pefformed ex vivo transfer of marker gene to heart grafts using adenovirus vector. Successful gene transfer and expression of beta-gal gene were demonstrated. This result demonstrated that cardiomyocytes of the grafts expressed an exogenous gene products by adenovirus mediated gene transfer under hypothermic preservation condition. 3) We found that gene gun- mediated transfer of the EBV-based episomal vector into rat hearts results in long-lasting expression of a marker gene. Combined use of the gene gun and EBV-based episomal vector may contribute to gene therapy of cardiovascular diseases. To our knowledge, this is the first study showing that in vivo gene transfer into heart can be achieved using a gene gun. 4) We transfected rodent cells with EBV-episomal vectors carrying E.coli b-galactosidase gene as a marker gene. Significantly higher transient expression was demonstrated, both in vivo and in vitro, in rodent cells transfected with the EBV vector compared to the cells transfected with a conventional vector. 5) We transplanted porcine cryopreserved aortic valve to dog aorta for the purpose of assessment of the ability of discordant xeno-tissue-transplantation. Compared with fresh tissue, cryopreserved valves kept cell viability. This result suggests the ability of clinical use.