Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.

中文翻译：

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通过克隆，改进了GTKO / hCD55 / hCD59三基因修饰的滇南微型猪的异种移植生产。

成功进行猪异种移植必须进行多种基因改造。然而，多基因修饰的细胞通常遭受各种药物选择和长期体外培养的困扰，这对于体细胞核移植（SCNT）生产转基因猪的性能较差。我们曾经通过使用SCNT的药物选择性细胞系来产生GTKO / hCD55 / hCD59三基因修饰猪，但是大多数克隆的猪都是转基因阴性个体。在这项研究中，为了提高多只转基因猪的生产效率，我们使用转基因猪胎儿成纤维细胞进行了克隆。结果，从12个活克隆胎儿中获得了两个表达hCD55和hCD59的胎儿，选择具有高转基因表达的一种作为供克隆的供体细胞来源。然后，我们获得了12只克隆的仔猪，全部是GTKO，带有hCD55和hCD59。hCD55和hCD59均在成纤维细胞中表达，但hCD55和hCD59的表达水平在这些仔猪之间不同。此外，仔猪P5＃在成纤维细胞中hCD55和hCD59的表达高于其他仔猪。相应地，小猪P5＃的成纤维细胞对人血清介导的细胞溶解的抵抗力明显高于小猪P11＃。总之，我们的研究结果首先为提高通过克隆产生多基因转基因猪的效率提供了支持。但这些仔猪中hCD55和hCD59的表达水平不同。此外，仔猪P5＃在成纤维细胞中hCD55和hCD59的表达高于其他仔猪。相应地，P5＃仔猪的成纤维细胞对人血清介导的细胞溶解的抵抗力明显高于P11＃仔猪。总之，我们的研究结果首先为提高通过克隆产生多基因转基因猪的效率提供了支持。但这些仔猪中hCD55和hCD59的表达水平不同。此外，仔猪P5＃在成纤维细胞中hCD55和hCD59的表达高于其他仔猪。相应地，P5＃仔猪的成纤维细胞对人血清介导的细胞溶解的抵抗力明显高于P11＃仔猪。总之，我们的研究结果首先为提高通过克隆产生多基因转基因猪的效率提供了支持。