Generation of a Fanconi anemia models in the pig by advanced genome engineering

在之前DFG资助的转座子介导的猪基因组转基因项目中，利用SB转座子系统建立了一种在猪中进行种系转基因和可靠的转基因表达的高效方法。在这里，我们建议通过使用转座子、Cre/loxP和簇状调节的间隔短回文重复序列(CRISPR/Cas9)系统在猪模型中建立一种有效的在卵基因组工程方法。拟议的实验将利用在上一个资助期建立的转座子转基因动物，为猪基因组的单步工程提供原则证明，目的是建立范科尼贫血的猪疾病模型。现有的范可尼贫血小鼠模型不能概括这种疾病的某些特征。预计Fanconi猪将反映人类的疾病表型，并将成为开发治疗方法的重要模式。拟议的项目是基于之前自己在建立转座子转基因以及在啮齿动物和猪身上产生诱导多能干细胞方面的专业知识。

In the previous DFG-funded project Transposon-mediated transgenesis in the pig genome a highly efficient approach for germline transgenesis coupled with reliable transgene expression in the pig was established employing the SB transposon system. Here, we propose to establish an efficient in ovo genome engineering approach in the pig model by employing transposon, Cre/loxP and clustered regulated interspaced short palindromic repeat (CRISPR/Cas9) systems. The proposed experiments will exploit the transposon-transgenic animals established in the previous funding period to provide proof of principle for single step engineering of the pig genome with the aim to generate a pig disease model for Fanconi anemia. The available mouse models for Fanconi anemia do not recapitulate certain features of the disease. It is anticipated that a Fanconi pig will mirror the disease phenotype seen in humans and will become an important model for the development of curative therapies. The proposed project is based on previous own expertise in establishing transposon transgenesis, as well as generation of induced pluripotent stem cells in rodents and pigs.