Division of Reproductive Engineering, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan.
Division of Reproductive Biotechnology and Innovation, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan.
Lab Anim (NY). 2022 Oct;51(10):256-274. doi: 10.1038/s41684-022-01053-5. Epub 2022 Oct 10.
Laboratory rats have been used in biomedical research for over 170 years. Recently, genome editing technology has facilitated the generation of genetically modified rats worldwide. This development has increased the demand for efficient preservation and production of rat resources. Sperm cryopreservation is the most efficient and robust means to archive genetic resources, and this technique reduces the number of animals required for colony management. Previously, we have reported a protocol for rat sperm cryopreservation and in vitro fertilization using frozen-thawed sperm. Here we describe an improved in vitro fertilization protocol to enhance the fertilization rate of cryopreserved sperm in major strains of rats. In this optimized protocol, treatment of frozen-thawed rat sperm with a high concentration of bovine serum albumin (40 mg/ml) results in a high in vitro fertilization rate. This protocol consists of three main steps: preparation of cryopreserved sperm, in vitro fertilization using cryopreserved sperm and embryo transfer. This process takes approximately 1 month to produce live pups from cryopreserved sperm. This protocol can be easily implemented by researchers and technicians with experience in reproductive engineering technology; it can also be used, albeit with some practice, by researchers and technicians who have no experience in reproductive techniques. This sperm cryopreservation and in vitro fertilization protocol for rats will provide an efficient system for the archiving and production of genetically modified rats for the transgenic community.
实验室大鼠在生物医学研究中已经使用了超过 170 年。最近，基因组编辑技术促进了世界各地转基因大鼠的产生。这一发展增加了对有效保存和生产大鼠资源的需求。精子冷冻保存是保存遗传资源最有效和最强大的手段，这项技术减少了对群体管理所需的动物数量。以前，我们已经报道了使用冷冻和解冻精子进行大鼠精子冷冻保存和体外受精的方案。在这里，我们描述了一种改进的体外受精方案，以提高主要大鼠品系中冷冻精子的受精率。在这个优化的方案中，用高浓度牛血清白蛋白（40mg/ml）处理冷冻解冻的大鼠精子，可获得高的体外受精率。该方案主要包括三个步骤：冷冻精子的制备、冷冻精子的体外受精和胚胎移植。从冷冻精子中产生活的幼仔大约需要 1 个月的时间。有生殖工程技术经验的研究人员和技术人员可以很容易地实施这个方案；对于没有生殖技术经验的研究人员和技术人员来说，虽然需要一些实践，但也可以使用这个方案。这个大鼠精子冷冻保存和体外受精方案将为转基因社区提供一个有效的遗传修饰大鼠的保存和生产系统。
