斑马鱼精子冻存与复苏方法与流程-遗传.PDFVIP

HEREDITAS (Beijing) 2012 9 , 34(9): 1211―1216 ISSN 0253-9772 实验指南 DOI: 10.3724/SP.J.1005.2012.01211 斑马鱼精子冻存与复苏实验方法与流程 郑乃中, 张博 , , 100871 斑马鱼是研究胚胎发育及其遗传机制的重要模式脊椎动物。目前人们已经积累了大量的突变体和转基因 鱼系, 如何安全、妥善地长期保存这些品系是每一个研究斑马鱼的实验室都会面临的问题。精子冻存与复苏技 术是目前最为简单有效的一种长期保存斑马鱼遗传品系的方法。应用这一技术不仅可以节省大量的鱼房空间与 人力、物力, 使鱼系的使用与保存更加灵活和持久, 更重要的是能够防止珍贵鱼系的意外丢失, 为鱼系保种提 供额外的保障。这类方法一般是通过体外挤出精子或研磨精巢获取新鲜的精子, 用适量的冻存液混匀后, 分装 保存在液氮罐中。需要时可以随时通过体外授精的方法使精子复苏。经过 30 年的发展, 随着冷冻保护剂的不 断改良和冻存与复苏条件的不断优化, 斑马鱼精子冻存与复苏技术已逐渐成熟。文章简单回顾了斑马鱼精子冻 存与复苏技术的历史与发展, 并重点介绍本实验室自 2005 年以来常规使用的斑马鱼精子冻存与复苏的方法及 具体流程。 斑马鱼; 精子冻存; 精子复苏; 体外授精 A brief protocol for sperm cryopreservation and revival in zebrafish ZHENG Nai-Zhong, ZHANG Bo Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China Abstract: Zebrafish is an important vertebrate model organism for the study of embryonic development and the under- lying genetic mechanism. Numerous mutants and transgenics have been generated in recent years, long-term and safe stor- age of these fish lines is of crucial importance for every zebrafish community/lab. Sperm cryopreservation and revival has become a preferred method for this purpose, which provides extra and reliable security as a backup for the cost-effective maintenance of genetic stocks in addition to reducing space demanding for housing large amount of live fish. This is espe- cially critical for invaluable fish lines against accidental loss. Generally, the sperm are obtained by either squeezing the male fish or dissecting out and homogenizing the testes, then they are mixed with the freezing medium before gradually frozen as aliquots in liquid nitrogen. They can be easily rev