HEREDITAS (Beijing) 2012 9, 34(9): 1211 1216 ISSN 0253-9772 www.chinagene.cn 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 underlying genetic mechanism. Numerous mutants and transgenics have been generated in recent years, long-term and safe storage 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 especially 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 revived through in vitro fertilization whenever necessary. This technique was 收稿日期 : 2012 08 31; 修回日期 : 2012 09 07 基金项目 : ( 31110103904) ( 2012CB945101) 作者简介 :,, Tel: 010-62753077; E-mail: peterz@pku.edu.cn 通讯作者 :,,, E-mail: bzhang@pku.edu.cn 致 谢 : NIH,,
1212 HEREDITAS (Beijing) 2012 34 introduced into zebrafish research three decades ago and has gradually become mature and more reliable following the improvement of many critical factors and steps, including cryoprotectants and conditions for freezing and revival. Base on pioneers work, our lab has established and improved a simple method for sperm cryopreservation and revival which shows high recovery rate after relatively long storage time. Here we briefly summarize the history and development of the methods for sperm cryopreservation and revival in zebrafish and present a brief protocol for the practice of sperm cryopreservation and revival, which is routinely used in our lab. Keywords: zebrafish; sperm cryo-preservation; sperm revival; in vitro fertilization (Danio rerio),,,, 30, Harvey [1],, 21, Morris [2],, ;,,,, (DMSO) (DMA N, N-dimethyl acetamide), 10% DMA [2], Draper Yang Carmichael, [3~6] Westerfield(The Zebrafish Book) [7] Sood [8] 鳉 (Medaka), N, N- (DMF N, N-dimethylformamide),, 2005 Sood [8], [9], ( 6 88%, 85% ) ( 5 ) 1 1.1 1.1.1 精子冻存用试剂 DMF(N, N-dimethylformamide)(Sigma, Cat 27054-7) Tricaine(Sigma, Cat A5040) Tricaine methanesulfonate, MS-222 3-aminobenzoic acid ethyl ester, ethyl m-aminobenzoate Tricaine, Tris; (Fetal bovine serum); (95%); ; 1.1.2 精子冻存用溶液 Tricaine (25 ) Tricaine 20,, Tricaine 400 mg; 97.9 ml; 1 mol/l Tris(pH 9)~2.1 ml, ph ~7
9 : 1213 Tricaine 1 4.2 ml Tricaine, 100 ml 10% DMF 1.1.3 精子冻存用器材与耗材 ; ( Dumont 5); (90 mm); ; ; ; ; (Axygen, ST-200-SS&SCO-A 2 ); ; (Harvard Apparatus, GC100-10 1.0 mm O.D. 0.58 mm I.D.,, ); ; (10 cm 2 ); ; Eppendorf (1.5 ml); Eppendorf ; ; ; ; 1.2 1.2.1 精子复苏用试剂 ( ) NaCl, KCl, CaCl 2, NaHCO 3, MgSO 4 7H 2 O, Na 2 HPO 4 ( ), KH 2 PO 4 ; ; 1.2.2 精子复苏用溶液 ; Hank s ( 4 ) #1 8.0 g NaCl 0.4 g KCl, 100 ml #2 0.358 g Na 2 HPO 4 ( ) 0.60 g KH 2 PO 4, 100 ml #4 0.72 g CaCl 2, 50 ml ; #5 1.23 g MgSO 4 7H 2 O, 50 ml ; #6 0.35 g NaHCO 3, 10 ml Hank s 10.0 ml #1 1.0 ml #2 1.0 ml #4 86.0 ml 1.0 ml #5, 4 (Hank s ) 9.9 ml Hank s, 0.1 ml #6, 4 1.2.3 精子复苏用器材与耗材 ; ; ; (90 mm); ; ; ; ; ; Eppendorf (1.5 ml); (1 ml 200 µl ); (1 ml 200 µl); ; ; 28.5 2 2.1,, 7~8,, 4, 10 20 min ( 1), 7 8,,, 6, 80% 15% 2.1.1 精子冻存准备工作 ( 冻存当日 ) (1) ( ) (1 kg 6 ), 95% ( ), (2), 1.5 ml Eppendorf, 80 90 μl(, ), (3) Tricaine Tricaine (4) ( ) 1 2 cm 2,, (, ) 2.1.2 取精巢及精子冻存操作 (1), Tricaine 2 3 min,,,,,, Tricaine (2) ( ),
1214 HEREDITAS (Beijing) 2012 34,, ( ),,,, ( 1, I) (3) ( ), Eppendorf,, (4) 7 8 Eppendorf,,, ; 3 4 ( )10 20 min,, 1 ( 1 2 1 2 ), 2.2 2.2.1 精子复苏准备工作,, ( ),, Hank s, 1.5 ml Eppendorf, 100 μl, 2.2.2 精子复苏操作, (1) ( ),, ( ),,, 图 1 解剖精巢冻存精子的主要步骤 A B Tricaine ; C D ; E F G ; I,,,, ; H J: Eppendorf ; K; L,, ; M 1 cm
9 : 1215 ( ), (2) Hank s ( ) Eppendorf, 200 μl,,,,, 200 μl ( ) 1 ml 1 ml (3) 2 min,, ( ) 28.5 30 min, 3 h, 1 000, 2 3,,,, 3 1,,, 90%,,, ( ),, 2 3,,,,,, 20 min, 2 4, 图 2 斑马鱼精子复苏和体外授精 A, ; B: ; C: Eppendorf ; D: ; E:,, ; F: ; G:, ; H: 2.5, 10 1 cm
1216 HEREDITAS (Beijing) 2012 34 4, 2 h 100, ( ) (0 ) ( 70 ) ( 196 ),, Tricaine 1,,, 5 Tricaine,, Tricaine,,,, ( 200 300 ),,,,,,,,,,,,,, 4,, 6 88%;, 3%, 6 ( ) 85% 7 8,, 85% 3 000, 400, 10, ( ),,,, 30%, 3%, 附录 (www.chinagene.cn) 参考文献 (References): [1] Harvey B, Ashwood-Smith MJ. Cryoprotectant penetration and supercooling in the eggs of salmonid fishes. Cryobiology, 1982, 19(1): 29 40. [2] Morris JPt, Berghmans S, Zahrieh D, Neuberg DS, Kanki JP, Look AT. Zebrafish sperm cryopreservation with N,Ndimethylacetamide. Biotechniques, 2003, 35(5): 956 958, 960, 962 passim. [3] Draper BW, Moens CB. A high-throughput method for zebrafish sperm cryopreservation and in vitro fertilization. J Vis Exp, 2009,(29):1395. [4] Yang H, Carmichael C, Varga ZM, Tiersch TR. Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio. Theriogenology, 2007, 68(2): 128 136. [5] Carmichael C, Westerfield M, Varga ZM. Cryopreservation and in vitro fertilization at the zebrafish international resource center. Methods Mol Biol, 2009, 546: 45 65. [6] Mazur P, Leibo SP, Seidel Jr GE. Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status, and future directions. Biol Reprod, 2008, 78(1): 2 12. [7] Westerfield M. The zebrafish book : a guide for the laboratory use of zebrafish (Brachydanio rerio). 1993, Eugene, OR: M. Westerfield. [8] Sood R, English MA, Jones M, Mullikin J, Wang DM, Anderson M, Wu D, Chandrasekharappa SC, Yu J, Zhang J, Paul Liu P. Methods for reverse genetic screening in zebrafish by resequencing and TILLING. Methods, 2006, 39(3): 220 227. [9] Wang D, Jao LE, Zheng N, Dolan K, Ivey J, Zonies S, Wu X, Wu K, Yang H, Meng Q, Zhu Z, Zhang B, Lin S, Burgess SM. Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. Proc Natl Acad Sci USA, 2007, 104(30): 12428 12433.