Attempt at creating donor pig by nuclear transplantation for xenotrasplantation

1)我和我的同事们曾试图创造出阿尔法-盖尔淘汰赛猪。现在，我们还没有成功。2)为了研究改变移植后同种或异种反应性的方法，我们利用腺病毒载体将标记基因体外转移到心脏移植物中。证实了β -半乳糖基因的成功转移和表达。结果表明，在低温保存条件下，移植物心肌细胞通过腺病毒介导的基因转移表达了外源基因产物。3)我们发现基因枪介导的以ebv为基础的episomal载体转移到大鼠心脏导致一个标记基因的长期表达。基因枪与基于ebv的episomal载体的联合应用可能有助于心血管疾病的基因治疗。据我们所知，这是第一次研究表明，体内基因转移到心脏可以实现使用基因枪。4)用携带大肠杆菌b-半乳糖苷酶基因作为标记基因的EBV-episomal载体转染啮齿动物细胞。在体内和体外，与传统载体转染的细胞相比，转染EBV载体的啮齿动物细胞的瞬时表达均显著提高。5)我们将猪低温保存的主动脉瓣移植到狗主动脉上，目的是评估不协调异种组织移植的能力。与新鲜组织相比，冷冻保存的瓣膜能保持细胞活力。这一结果提示了其临床应用的能力。

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.