鹅原始生殖细胞体外分离与冷冻保存方法的比较研究

doi:10.3969/j.issn.1004-1524.2017.03.08

浙江农业学报 ›› 2017, Vol. 29 ›› Issue (3): 401-407.DOI: 10.3969/j.issn.1004-1524.2017.03.08

鹅原始生殖细胞体外分离与冷冻保存方法的比较研究

于建宁, 闫乐艳, 陈哲, 邵西兵, 戴子淳, 应诗家, 施振旦^*

江苏省农业科学院畜牧研究所/动物品种改良和繁育重点实验室,江苏南京 210014

收稿日期:2016-11-23 出版日期:2017-03-20 发布日期:2017-03-31
作者简介:于建宁(1980—),女,山东烟台人,博士,副研究员,从事家禽生物技术研究。E-mail:jianningyu @aliyun.com
通讯作者: 施振旦,E-mail:zdshi@jaas.ac.cn
基金资助:
江苏省农业科技自主创新资金项目[CX(14)2068]; 江苏省自然科学基金(BK20130718)

Comparison of methods to isolate and cryopreserve goose primordial germ cells

YU Jianning, YAN Leyan, CHEN Zhe, SHAO Xibing, DAI Zichun, YING Shijia, SHI Zhendan^*

Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Laboratory of Animal Improvement and Reproduction, Nanjing 210014, China

Received:2016-11-23 Online:2017-03-20 Published:2017-03-31

摘要/Abstract

摘要： 为了建立针对鹅原始生殖细胞(primordial germ cells,PGCs)的分离纯化与冷冻保存方法,对血液Ficoll密度梯度离心法、血液ACK裂解法、生殖腺胰酶-EDTA消化法与生殖腺消化结合体外短时间培养法4种PGCs分离方法以及STEM-CELLBANKER、CELLBANKER 2与10%DMSO加90%血清等3种冻存液进行了比较研究。通过形态学观察、糖原染色与PGCs特异免疫荧光染色鉴定PGCs,同时利用台盼蓝染色检测PGCs活力等方法对其分离和保存效果进行评价。结果表明,从鹅胚血液与生殖腺中均已经成功分离到鹅PGCs;生殖腺消化结合体外短期培养法与传统的血液分离法相比,获得的鹅PGCs数量、纯度与活力最高,每枚胚胎获得的鹅PGCs数量达到115个、细胞纯度达80.8%、细胞活率达88.3%;而商业化的干细胞冷冻保护液STEM-CELLBANKER同一般的体细胞冻存液相比更适合鹅PGCs的冷冻保存,冷冻解冻后的细胞存活率达90%左右。试验可以得出结论:生殖腺消化结合体外短期培养法成功分离到数量与纯度都可观的鹅PGCs,STEM-CELLBANKER冻存液能够用于鹅PGCs的长期冷冻保存。

关键词: 鹅, 原始生殖细胞, 生殖腺消化结合短期培养法, 冷冻保存

Abstract: In order to establish the isolation and cryopreservation method for goose primordial germ cells (PGCs), four kinds of PGCs separation methods,which were blood Ficoll density gradient centrifugation, blood ACK lysis method, gonads trypsin-EDTA digestion method and gonads digestion combined with short-term culture method, were compared in this experiment. And three kinds of cryoprotectors that were STEM-CELLBANKER, CELLBANKER 2 and 10% DMSO plus 90% serum were compared to evaluate the cryopreservation efficiency of goose PGCs. Morphological observation, PAS and PGCs specific immunofluorescence staining were used to verify PGCs, and trypan blue staining was used to detect the vitality of PGCs. The results showed that goose PGCs were successfully isolated from the blood and gonad of goose embryo. Gonads digestion combined with short-term culture method got the highest goose PGCs quantity, purity and vitality, reached 115 cells per embryo, 80.8% and 88.3% respectively, compared with the traditional blood separation method. Commercial stem-cell cryoprotector STEM-CELLBANKER was more suitable for cryopreservation of goose PGCs than the normal cell cryoprotectants, and the cell survival rate was about 90% after thawing. Gonads digestion combined with short-term culture method could get sufficient quantity and purity of goose PGCs. STEM-CELLBANKER could be used for the long-term cryopreservation of goose PGCs.

Key words: goose, primordial germ cells, gonads digestion combined with short-term culture, cryopreservation

中图分类号:

S835

于建宁, 闫乐艳, 陈哲, 邵西兵, 戴子淳, 应诗家, 施振旦. 鹅原始生殖细胞体外分离与冷冻保存方法的比较研究[J]. 浙江农业学报, 2017, 29(3): 401-407.

YU Jianning, YAN Leyan, CHEN Zhe, SHAO Xibing, DAI Zichun, YING Shijia, SHI Zhendan. Comparison of methods to isolate and cryopreserve goose primordial germ cells[J]. , 2017, 29(3): 401-407.

参考文献

[1] 汤青萍,章双杰,郭军,等. 太湖鹅群体遗传多样性研究[J]. 家畜生态学报,2010,31(1):30-33.
TANG Q P, ZHANG S J, GUO J, et al. Analysis of genetic diversity in Taihu geese[J]. Journal of Domestic Animal Ecology , 2010, 31(1):30-33. (in Chinese with English abstract)
[2] 吴常信. 畜禽遗传资源保存的理论与技术[J]. 家畜生态,2001,22 (1):1-4.
WU C X. Theory and technology of preservation in domestic animal genetic resources[J]. Ecology of Domestic Animal , 2001, 22 (1):1-4. (in Chinese with English abstract)
[3] NAKAMURA Y, TASAI M, TAKEDA K, et al. Production of functional gametes from cryopreserved primordial germ cells of the Japanese quail[J]. Journal of Reproduction and Development , 2013, 59(6):580-587.
[4] NAKAMURA Y, USUI F, MIYAHARA D, et al. Efficient system for preservation and regeneration of genetic resources in chicken: concurrent storage of primordial germ cells and live animals from early embryos of a rare indigenous fowl (Gifujidori)[J]. Reproduction Fertility and Development , 2010, 22(8):1237-1246.
[5] NAKAMURA Y, KAGAMI H, TAGAMI T. Development, differentiation and manipulation of chicken germ cells[J]. Development , Growth and Differentiation , 2013, 55(1):20-40.
[6] 韩毅冰,于康震,周琦,等. 鸡胚血液中原始生殖细胞的分离及其培养的研究[J]. 生物技术,1996,6(2):11-13.
HAN Y B, YU K Z, ZHOU Q, et al. Studies on the isolation and culture of primordial germ cells from blood of chicken embryos[J]. Biotechnology , 1996, 6(2):11-13. (in Chinese)
[7] SONG Y, DURAISAMY S, ALI J, et al. Characteristics of long-term cultures of avian primordial germ cells and gonocytes[J]. Biology of Reproduction , 2014, 90(1):15.
[8] 李碧春,秦洁,肖小琚,等. 鸡胚不同发育时期原始生殖细胞的分离方法[J]. 动物学报,2003,49(6):835-842.
LI B C, QIN J, XIAO X X, et al. A method for isolation of primordial germ cells in different stages of the chicken embryo[J]. Acta Zoologica Sinica , 2003, 49(6):835-842. (in Chinese with English abstract)
[9] YAMAMOTO Y, USUI F, NAKAMURA Y, et al. A novel method to isolate primordial germ cells and its use for the generation of germline chimeras in chicken[J]. Biology of Reproduction , 2007, 77(1): 115-119.
[10] 李赞东,刘春海,黄劲松,等. 鸡鸭异种间嵌合体的制备[J]. 动物学报,2002,48(4):543-548.
LI Z D, LIU C H, HUANG J S, et al. Preparation of Interspecies chicken ( Gallus domesticus )/duck ( Anas domesticus ) chimeras[J]. Acta Zoologica Sinica , 2002, 48(4):543-548. (in Chinese with English abstract)
[11] ZHANG W, RUI L, ZHANG J, et al. Production of chimeras between the Chinese soft-shelled turtle and Peking duck through transfer of early blastoderm cells[J]. Journal of Experimental Biology , 2013, 216(Pt 10):1786-1792.
[12] YAN L, GAO J S, RUI L, et al. A review of strategies for producing chimeric birds[J]. Avian Biology Research , 2014, 7(1):3-9.
[13] 孙鹏翔. 鸡胚原始生殖细胞体外转染及其介导的转基因研究[D]. 扬州:扬州大学,2007:19-32.
SUN P X. Study on the in vitro transfection and transgenic with embryonic primordial germ cells [D]. Yangzhou: Yangzhou University, 2007:19-32. (in Chinese with English abstract)
[14] 唐新燕,张才乔. 鸡胚原始生殖细胞体外的分化[J]. 农业生物技术学报,2007,15(1):20-23.
TANG X Y, ZHANG C Q. Differentiation of chicken primordial germ cells in vitro[J]. Journal of Agricultural Biotechnology , 2007, 15 (1):20-23. (in Chinese with English abstract)
[15] 葛楚天. 鸡胚原始生殖细胞发育调控及其机理的研究[D]. 杭州:浙江大学,2010:38.
GE C T. Regulation of the chicken primordial germ cell development [D]. Hangzhou: Zhejiang University, 2010:38. (in Chinese with English abstract)

鹅原始生殖细胞体外分离与冷冻保存方法的比较研究

Comparison of methods to isolate and cryopreserve goose primordial germ cells

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	贾北平, 吕炫, 杨庆, 王一楠, 李皖萧, 解新迪, 朱英奇, 王蓓, 殷冬冬, 张云凯, 王晴, 王桂军. 安徽省5株新型鹅星状病毒的分离鉴定与遗传进化分析[J]. 浙江农业学报, 2023, 35(5): 1048-1057.
[2]	刘林雨, 张扬, 刘威, 包强, 翁恺麒, 徐欣磊, 张钰, 徐琪, 陈国宏. 国内外5个典型鹅品种线粒体DNA遗传多样性与系统进化分析[J]. 浙江农业学报, 2023, 35(11): 2525-2532.
[3]	李虹仪, 周润盛, 梁笑玲, 张楚玥, 吕祺欣, 杨长华, 张茂. 日粮钙磷水平对马岗鹅生长性能及肝脏基因表达的影响[J]. 浙江农业学报, 2023, 35(11): 2533-2542.
[4]	朱寅初, 王宏宇, 云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备[J]. 浙江农业学报, 2022, 34(10): 2149-2159.
[5]	王志, 胡仲皓, 桂雪儿, 冯士彬, 李玉, 王希春, 李锦春, 吴金节. 饲喂高蛋白日粮对雏鹅血清尿酸水平、肝脏和肾脏超微结构及ABCG2表达的影响[J]. 浙江农业学报, 2021, 33(5): 801-808.
[6]	蒲路莎, 苏世博, 陈肖韩, 赵丽丽, 陈洪岩. 鹅源星状病毒ORF2基因原核表达及遗传进化分析[J]. 浙江农业学报, 2020, 32(5): 789-797.
[7]	王淑娟, 刘文举, 闻爱友, 庞训胜. 鹅褪黑素受体Mel1c基因的组织表达特征[J]. 浙江农业学报, 2018, 30(9): 1454-1459.
[8]	陈兵, 徐婧, 罗启慧, 黄超, 刘文涛, 吴洪, 陈镜妃, 陈苹, 高琪, 马文静, 陈正礼. 感染鸭源小鹅瘟病毒QH-L01株樱桃谷雏鸭病理学研究[J]. 浙江农业学报, 2018, 30(6): 932-938.
[9]	张扬, 王美青, 穆春宇, 姚文成, 杨耀宗, 卞友庆, 陈国宏. 鹅线粒体COⅠ条形码及上游tRNA序列测定分析[J]. 浙江农业学报, 2017, 29(1): 51-57.
[10]	杨泽晓1，2，刘常钰1，王印1，2，姚学萍1，邬旭龙1，王波1，李桂黎1，蒙正群1，刘亚东1. 一株鹅源多杀性巴氏杆菌强毒株的分离鉴定及其血清型鉴定相关基因的克隆[J]. 浙江农业学报, 2016, 28(6): 935-.
[11]	徐小钦1，2，陈黎1，李进军1，杜雪1，田勇1，卢立志1，2，*. 微卫星标记技术鉴定某保种场四川白鹅保种效果 [J]. 浙江农业学报, 2015, 27(5): 751-.
[12]	韩凯凯，李银*，黄欣梅，赵冬敏，刘宇卓，刘青涛，谢星星，安凤娇. 鹅坦布苏病毒非结构蛋白NS5的原核表达及特性分析[J]. 浙江农业学报, 2015, 27(10): 1704-.
[13]	麻延峰1，方倩倩2，3，王宏艳1，田勇2，唐军旺2，邵荣益4，卢立志2. 鹅Apo A1，CD36和DGAT2基因的cDNA克隆与序列分析[J]. 浙江农业学报, 2015, 27(10): 1710-.
[14]	王健1，董飚1，2，侯庆永3，殷洁鑫1. 太湖鹅MyoG基因多态性与体重的相关性分析[J]. 浙江农业学报, 2015, 27(1): 28-.
[15]	韩凯凯;李银*;黄欣梅;赵冬敏;刘宇卓;谢星星;游园. 鹅源坦布苏病毒RT\\|LAMP快速检测方法的建立[J]. , 2014, 26(1): 0-33.