C57BL/6J背景基因修饰小鼠精子冷冻保种及品系恢复方法

doi:10.3969/j.issn.1674-5817.2019.02.014

实验动物与比较医学 ›› 2019, Vol. 39 ›› Issue (2): 146-150.DOI: 10.3969/j.issn.1674-5817.2019.02.014

C57BL/6J背景基因修饰小鼠精子冷冻保种及品系恢复方法

牛博文, 陈丽香, 朱孟敏, 彭秀华, 秦波音, 李峰

上海市公共卫生临床中心, 上海 201508

收稿日期:2018-10-15 出版日期:2019-04-25 发布日期:2021-01-29
作者简介:牛博文(1990-),男,助理兽医师,主要从事基因修饰动物模型研究,E-mail:goodniubowen@163.com
基金资助:
上海市公共卫生临床中心院内课题项目(KY-GW- 2017-04, KY-GW-2017-06,KY-GW-2018-11), 上海市高等级生物安全病原微生物检测专业技术服务平台建设项目(18DZ2293000)

Sperm Cryopreservation and Recovery of C57BL/6J Background Genetically Modified Mice

NIU Bo-wen, CHEN Li-xiang, ZHU Meng-min, PENG Xiu-hua, QIN Bo-yin, LI Feng

Shanghai Public Health Clinical Center, Shanghai 201508, China

Received:2018-10-15 Online:2019-04-25 Published:2021-01-29

摘要/Abstract

摘要： 目的建立一种高效的C57BL/6J背景基因修饰小鼠的精子冷冻及体外受精（IVF）方法。方法以C57BL/6J小鼠为研究对象,围绕冷冻保护液、冷冻精子浓度、复苏后精子处理方法及IVF培养液等方面进行研究,IVF后2-细胞发育率作为冷冻和复苏效率评价标准,对比各组之间发育情况。结果以R18S3(含质量比为18%棉子糖和3%脱脂奶粉)、M-R18S3冷冻保护液[含终浓度为477 µmol/L硫代甘油(MTG)的R18S3冷冻液]进行精子冷冻,复苏后在HTF培养液中进行IVF,2-细胞发育率分别为8.4%和20.6%; 以R18S3和M-R18S3作为冷冻保护液,以含浓度为1 mmol/L还原性谷胱甘肽(GSH)的HTF培养液进行IVF,2-细胞发育率分别为25.0%和43.5%。高浓度精子冷冻及复苏法可获得64.2%的2-细胞发育率; 选取4种C57BL/6J 背景基因修饰小鼠进行精子冷冻及IVF,2-细胞发育率34%~90%,胚胎移植后出生率40%~57%。结论 M-R18S3作为冷冻保护液,采用高浓度精子冷冻法进行精子冷冻,冷冻精子复苏后进行受精滴洗涤筛选,以含1 mmol/L GSH的HTF培养液进行冷冻精子IVF,此技术体系可以作为C57BL/6J背景基因修饰鼠的保种及品系恢复手段。

关键词: 精子冷冻, 体外受精(IVF), 硫代甘油(MTG), 谷胱甘肽(GSH), C57BL/6J小鼠

Abstract: Objective To establish a high-efficiency sperm cryopreservation and in vitro fertilization (IVF) method on C57BL/6J background gene-modified mice. Methods This study compared the effects of different methods on sperm cryoprotectant, sperm concentration, treatment after thawing and IVF medium on 2-cell cleavage rate, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6J backgrounds. Results Sperm were frozen with R18S3, which containing 18% gossylose and 3% skimmed milk powder, then thawed for IVF in HTF medium and their fertility (two-cell cleavage) was 8.4%. Sperm were frozen with M-R18S3, a cryoprotective solution that containing 477 µmol/L monothioglycerol (MTG) in R18S3 and then IVF in HTF medium, their fertility (two-cell cleavage) was 20.6%. Sperm cryopreserved in M-R18S3, IVF in HTF with 1mmol/L reduced glutathione (GSH), have better IVF rate than when cryopreserved in R18S3 alone (43.5% vs 25%). High concentration sperm was cryopreserved in M-R18S3, then thawed for IVF in HTF medium with 1 mmol/L GSH and their fertility (two-cell cleavage) was higher than low concentration sperm (64.2% vs 43.5%). Sperm from 3 knockout and 1 transgenic mouse lines on C57BL/6J backgrounds cryopreserved using this techniques were all thawed successfully to obtain IVF fertility (two-cell cleavage) between 34% and 90%, and after embryo transplantation to get genotypically confirmed offspring. Conclusion High concentration sperm was frozen in M-R18S3, screened in IVF drop after thawing, and then IVF in HTF with 1 mmol/LGSH, can be used as an effective method for sperm preservation and recovery of C57BL/6J background genome-modified mice.

Key words: Sperm cryopreservation, In vitro fertilization (IVF), Monothioglycerol (MTG)

中图分类号:

Q95-33

牛博文, 陈丽香, 朱孟敏, 彭秀华, 秦波音, 李峰. C57BL/6J背景基因修饰小鼠精子冷冻保种及品系恢复方法[J]. 实验动物与比较医学, 2019, 39(2): 146-150.

NIU Bo-wen, CHEN Li-xiang, ZHU Meng-min, PENG Xiu-hua, QIN Bo-yin, LI Feng. Sperm Cryopreservation and Recovery of C57BL/6J Background Genetically Modified Mice[J]. Laboratory Animal and Comparative Medicine, 2019, 39(2): 146-150.

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C57BL/6J背景基因修饰小鼠精子冷冻保种及品系恢复方法

Sperm Cryopreservation and Recovery of C57BL/6J Background Genetically Modified Mice

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