Hirabayashi Masumi, Hochi Shinichi
The Graduate University for Advanced Studies, Okazaki, Aichi, Japan.
Section of Mammalian Transgenesis, Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.
Methods Mol Biol. 2019;1874:313-326. doi: 10.1007/978-1-4939-8831-0_18.
Regeneration of human organs in domestic animal model would provide enough number of functional donor organs in transplantation therapy. Recent progresses in pluripotent stem cells and nuclease-based genome editing tools have set the stage for investigating the chimeric complementation approach to generate functional organs from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. In this chapter, protocol for allogeneic or xenogeneic organ generation using knocked-out (KO) rat blastocysts and the rat or mouse ES/iPS cells is described. The protocol includes (1) the preparation of KO rat colony, (2) the preparation of rat or mouse ES/iPS cells, (3) the recovery of rat blastocysts, (4) the stem cell injection into blastocysts, (5) the embryo transfer into pseudopregnant recipient uteri, and (6) the genotyping and organogenetic analysis of chimeric offspring. The accumulation of basic and practical knowledge in the rodent model would be useful in improving therapeutic performance to regenerate 3D organs available for transplantation.
在家畜模型中实现人类器官再生将为移植治疗提供足够数量的功能性供体器官。多能干细胞和基于核酸酶的基因组编辑工具的最新进展为研究嵌合互补方法奠定了基础，该方法可从胚胎干细胞（ES细胞）或诱导多能干细胞（iPS细胞）生成功能性器官。在本章中，将描述使用基因敲除（KO）大鼠囊胚以及大鼠或小鼠ES/iPS细胞生成同种异体或异种器官的方案。该方案包括：（1）制备基因敲除大鼠群体；（2）制备大鼠或小鼠ES/iPS细胞；（3）回收大鼠囊胚；（4）将干细胞注入囊胚；（5）将胚胎移植到假孕受体子宫；（6）对嵌合后代进行基因分型和器官发生分析。啮齿动物模型中基础和实践知识的积累将有助于提高再生可用于移植的3D器官的治疗性能。
