Takizawa Akiko, Eto Tomoo
Department of Physiology, Genomic Sciences and Precision Medicine Center, Medical College of Wisconsin, Milwaukee, WI, USA.
Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan.
Methods Mol Biol. 2019;2018:131-149. doi: 10.1007/978-1-4939-9581-3_6.
New genome-editing tools, such as ZFNs, TALEN, and CRISPR/Cas9, have enabled the generation of gene-modified models effectively in mammals. These technologies are a powerful tool for studying gene function and creating animal models for human diseases. On the other hand, such gene-modified animals are raised in numerous experimental animal facilities, which puts pressure on breeding space and maintenance costs. Embryo and sperm cryopreservation is not only the most simple and cost-effective method available for most gene-modified strains but also the most reliable method to preserve strains to avoid breeding problems and contamination. We have established a reliable, high quality embryo and sperm cryopreservation system for rat strains, ensuring the longevity of these valuable resources for the scientific community. These cryopreserved resources have been successfully used to rederive next generation pups using embryo transfer and intracytoplasmic sperm injection (ICSI). In this chapter, we describe in detail protocols for rat embryo vitrification and sperm cryopreservation followed by pup rederivation using the ICSI procedure and embryo transfer.
新型基因组编辑工具，如锌指核酸酶（ZFNs）、转录激活样效应因子核酸酶（TALEN）和规律成簇间隔短回文重复序列/Cas9（CRISPR/Cas9），已能够在哺乳动物中有效地生成基因修饰模型。这些技术是研究基因功能以及创建人类疾病动物模型的有力工具。另一方面，此类基因修饰动物在众多实验动物设施中饲养，这给繁殖空间和饲养成本带来了压力。胚胎和精子冷冻保存不仅是适用于大多数基因修饰品系的最简单且最具成本效益的方法，也是保存品系以避免繁殖问题和污染的最可靠方法。我们已经为大鼠品系建立了一个可靠、高质量的胚胎和精子冷冻保存系统，确保这些宝贵资源能长期为科学界所用。这些冷冻保存的资源已成功用于通过胚胎移植和胞浆内精子注射（ICSI）来繁育下一代幼崽。在本章中，我们将详细描述大鼠胚胎玻璃化冷冻和精子冷冻保存的方案，以及随后使用ICSI程序和胚胎移植来繁育幼崽的过程。
