戴尅戎Cell Death Differ新突破：首次利用精原干细胞获转基因树鼩

研究人员首次建立的树鼩精原干细胞体外培养体系，并利用体外培养的树鼩精原干细胞成功获得世界首只转基因树鼩，实现了树鼩基因修饰技术的重大突破。

　　

  12月23日，《细胞研究》（Cell Research）期刊在线发表了中国科学院昆明动物研究所郑萍课题组、赵旭东课题组和姚永刚课题组共同完成的题为Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring的研究论文，该文报道了他们首次建立的树鼩精原干细胞体外培养体系，并利用体外培养的树鼩精原干细胞成功获得世界首只转基因树鼩，实现了树鼩基因修饰技术的重大突破。

　　树鼩是一种具有重要潜在应用价值的实验动物。相较于啮齿类实验动物，树鼩在遗传进化地位及生理生化特征上均更接近于灵长类。并且，树鼩相比于灵长类动物在生物医学研究中有着诸多优势，包括体型小、生殖周期短、饲养成本低等。然而，缺乏遗传操作手段严重制约了树鼩作为实验动物的广泛应用和推广。由于树鼩早期胚胎收集和胚胎移植技术的难题目前均未解决，通过树鼩精原干细胞进行遗传修饰，可为树鼩遗传操作另辟蹊径。

　　在昆明动物所研究员郑萍等的指导下，博士研究生李朝晖等首先成功筛选到细胞表面标记分子Thy1可用于富集树鼩睾丸中的精原干细胞，并发现Wnt/ß-catenin信号通路及树鼩睾丸支持细胞对于精原干细胞维持其增殖及干细胞特征至关重要，据此改善了树鼩精原干细胞体外扩增培养体系，并建立了多株可长期稳定传代培养的树鼩精原干细胞系（图A）。这些体外长期培养的树鼩精原干细胞可稳定表达外源基因（图B），也可以利用CRISPR-Cas9进行基因编辑。基因修饰后的树鼩精原干细胞移植到经白消安处理的雄性受体树鼩睾丸内，可以产生基因修饰精子，通过自然交配从而获得基因修饰子代树鼩（图C、D）。

　　该工作解决了树鼩研究中缺乏基因操作技术的瓶颈难题，为树鼩作为新型实验动物的广泛应用打下良好基础。李朝晖为论文第一作者，郑萍、赵旭东和姚永刚为共同通讯作者。该工作得到了中科院（XDB13000000 and XDA01010203）、云南省（2015HA038）以及昆明动物所“135”项目资助。

 

　　A. 培养40代的树鼩精原干细胞系；B. 稳定表达绿色荧光蛋白（GFP）的树鼩精原干细胞；C. 基因修饰精原干细胞移植到受体睾丸中；D. 左边为表达GFP的转基因树鼩。

原文摘要：

Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/β-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.