GENE MODIFIED CLONED PIGS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该提案的最终目标是通过改变猪的基因来缓解器官短缺危机，这样猪的组织和器官就可以移植到人类身上（即异种移植），其结果与传统免疫抑制下的同种异体移植相同。这一目标的进展一直被高等灵长类受体的先天免疫反应所阻碍，这些免疫反应针对的是在猪细胞表面表达的alphaGal表位，而不是人类细胞。在研究了几种低等哺乳动物（包括猪）和所有高等哺乳动物（包括人类）的alphaGT基因的全基因组组织和转录调控后，我们正与PPL Therapeutics合作，首次产生具有双alpha1,3gt等位基因敲除（KO）的重组猪细胞。这些双KO细胞可用于体细胞核移植（克隆）产生克隆的双KO胎儿和仔猪。我们打算充分表征重组胎儿细胞的分子改变和表型质量，以及预期的足月克隆仔猪的细胞、组织或器官。此外，双KO胎儿细胞以及这些仔猪的细胞、组织和器官将在转基因小鼠模型中进行测试，以预测α gal阴性的高等灵长类受体（包括人类）对转基因猪异种移植物产生的先天和适应性免疫反应。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The ultimate objective of this proposal is to relieve the organ shortage crisis by genetic alteration of pigs so porcine tissues and organs can be transplanted to humans (i.e. xenotransplantation) with results equivalent to those attainable with allografts under conventional immunosuppression. Progress toward this objective has been precluded by the innate immune reaction of higher primate recipients that targets the alphaGal epitopes that are expressed on the surface of pig but not human cells. Having characterized the full genomic organization and transcriptional regulation of the alphaGT gene in several lower mammals (including pigs) and in all of the higher mammals (including humans), we are collaborating with PPL Therapeutics in their efforts to generate for the first time recombinant pig cells that have double alpha1,3GT allele knockout (KO). These double KO cells can be used for somatic cell nuclear transfer (cloning) to produce cloned double KO fetuses and piglets. We intend to fully characterize the molecular alterations and phenotypic qualities of the recombinant fetal cells, as well as the cells, tissues, or organs of the anticipated full-term cloned piglets. In addition, the double KO fetal cells, and the cells, tissues, and organs of these piglets will be tested in transgenic mouse models that will allow prediction of the innate and adaptive immune responses generated by alphaGal-negative higher primate recipients (including humans) to the genetically altered porcine xenografts.