,中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,国家斑马鱼资源中心,武汉 430072Development of resources, technologies and services at the China Zebrafish Resource Center
Feng Xiong, Xunwei Xie, Luyuan Pan, Kuoyu Li, Liyue Liu, Yun Zhang, Linglu Li, Yonghua Sun,China Zebrafish Resource Center, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China通讯作者:
编委: 刘峰
收稿日期:2018-05-29修回日期:2018-07-11网络出版日期:2018-08-16
| 基金资助: |
Received:2018-05-29Revised:2018-07-11Online:2018-08-16
| Fund supported: |
作者简介 About authors
熊凤,博士,工程师,研究方向：斑马鱼模式动物E-mail:
摘要
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熊凤, 谢训卫, 潘鲁媛, 李阔宇, 柳力月, 张昀, 李玲璐, 孙永华. 国家斑马鱼资源中心的资源、技术和服务建设[J]. 遗传, 2018, 40(8): 683-692 doi:10.16288/j.yczz.18-151
Feng Xiong, Xunwei Xie, Luyuan Pan, Kuoyu Li, Liyue Liu, Yun Zhang, Linglu Li, Yonghua Sun.
斑马鱼(Danio rerio)作为模式动物在生命科学、健康科学和环境科学等研究领域中扮演着越来越重要的角色。其基因组序列和人类的相似度达到70%以上[1],素有“水中小白鼠”之称。斑马鱼具有产卵量大、发育迅速、胚胎透明、体外受精和体外发育等生物学特征,使得研究者可以更加高效地开展各类实验[2,3]。
利用PubMed数据库统计发现,在2010~2017年间以斑马鱼为材料发表的研究论文增加速度明显高于小鼠(Mus musculus)、果蝇(Drosophila melanogaster)和线虫(Caenorhabditis elegans)等6种实验动物的研究(图1A)。在中国,自1998年第一家斑马鱼实验室在清华大学建立以来,斑马鱼研究群体迅速发展,发表的论文数量逐年递增[4]。2010年,中国发表的斑马鱼相关论文在国际论文中占比首次超过10%[5],中国成为仅次于美国的第二大斑马鱼研究大国。此后,我国以斑马鱼为研究材料发表的科研论文总数占全球同类研究的比重一直保持快速增加的势头(图1B)。
图1
新窗口打开|下载原图ZIP|生成PPT图1统计分析以斑马鱼为研究材料发表的科研论文
A:各主要实验动物发表相关论文在2010~2017年的发展趋势图。通过PubMed数据库以各类实验动物为主题分析出其相关论文数量,X轴是时间,Y轴是各个实验动物在各时期发表论文数占该实验动物在8年中论文总数的比例;B:全球主要斑马鱼研究国家发表斑马鱼相关论文占全球斑马鱼论文比例在2010~2017年的发展趋势图。通过Web of Science数据库以斑马鱼为主题同时限定区域分析出6个主要研究大国以斑马鱼为研究材料发表的科研论文数量。X轴是时间,Y轴是各国论文数占国际论文总数比例。
Fig. 1Statistical analysis of scientific papers related to zebrafish research
为加强国内斑马鱼研究人员的交流,中国自2010年开始举办每年一届的全国性斑马鱼大会。2012年以后,全国斑马鱼PI(Principal Investigator)大会和全国斑马鱼研究大会交替举行成为业界瞩目的盛会。2012年,首届中国斑马鱼PI大会的参会人数仅80人;而到了2016年,前来参加第三届中国斑马鱼PI大会的人数达到空前的240人;全国斑马鱼研究大会的参会人数也呈现爆发式增长,从2010年的50人迅速增加到2017年的600人。随着我国斑马鱼研究的迅速发展和研究团队的迅速扩张,迫切需要建设国家级的斑马鱼资源中心,以便更加高效地收集、创制、保藏和分享各类斑马鱼研究资源。
中国科学院水生生物研究所(以下简称水生所)是我国淡水鱼类生物学研究的综合性研究机构,在这里诞生了世界首例转基因鱼和体细胞克隆鱼[6]。鉴于此,在2010年4月举行的“第一届全国斑马鱼研究大会”上,朱作言院士和孟安明院士等40多位研究****一致呼吁,尽快在水生所建立“国家斑马鱼资源中心”,将我国****已有的斑马鱼品系和相关研究资源收集、保藏和分享,以提高科研效率和促进各单位间的合作。2012年10月,国家斑马鱼资源中心(China Zebrafish Resource Center, CZRC)在水生所正式挂牌成立,这对我国的斑马鱼研究具有标志性意义[7]。本文将对CZRC的资源建设情况以及对外服务等工作内容进行简要介绍,以增强研究者和公众对CZRC的了解。
1 硬件设施建设
2011年初,水生所启动了CZRC一期建设,至2014年,CZRC二期建设完成。CZRC的建设面积为600 m2,由一流标准的斑马鱼资源保藏区和遗传学实验室组成。其中,资源保藏区包括主鱼房、外来鱼隔离房、育苗室、显微注射室和精子超低温保藏室等功能区域(图2)。主鱼房装配有6套全自动水循环养殖系统,共计72个斑马鱼养殖架,可活体养殖16万条成年斑马鱼;外来鱼隔离房装配有4个独立水循环养殖架,可平行引进400多个斑马鱼品系;显微注射室装配有6套显微注射仪,可日均注射数以万计的斑马鱼胚胎;精子超低温保藏室装配有程序降温仪和2个600 L大型气态液氮罐,可开展高通量精子冻存工作,容纳5万管以上的精子冻存样本。遗传学实验室配备有开展各类分子生物学、细胞生物学、组织学和生物影像学等研究的仪器设备,可进行品系研制、基因型鉴定、表型分析和病理诊断等相关实验研究。先进的斑马鱼养殖设备和完善的分子生物学实验条件为CZRC的工作开展提供了有力的保障。图2
新窗口打开|下载原图ZIP|生成PPT图2CZRC鱼房鸟瞰效果图
Fig. 2Aerial view of the CZRC fish facility
2 资源建设与服务
2.1 资源建设及保藏
各类转基因和突变品系资源、基因资源等是利用斑马鱼开展发育生物学、遗传学、生物医学等研究的重要工具[8, 9]。CZRC通过资源交换、引进和创建,已保藏超过1200个斑马鱼品系(图3A),超过国际斑马鱼资源中心(Zebrafish International Resource Center, ZIRC)建设前10年的品系资源增量。此外,CZRC还保藏有2000多个DNA探针、150个质粒资源、61个斑马鱼抗体资源和2个斑马鱼细胞系等。所有的资源信息均公布在国家斑马鱼资源中心网站(http://zfish.cn),并可以通过国际斑马鱼信息中心数据库(http://zfin.org)进行交叉搜索。图3
新窗口打开|下载原图ZIP|生成PPT图3CZRC品系资源保存与服务
A:CZRC成立前5年的品系资源库增量情况(蓝色堆积曲面图,左坐标轴)和每年的资源服务人次增量情况(紫色柱状图,右坐标轴)。B:CZRC精子冷冻库质量评估。散点图表示单次操作的复苏受精率,红色折线为同批次冻存样品的复苏受精率平均值。
Fig. 3Resources and services in CZRC
从创建初始,CZRC积极与国内外科研人员开展合作,引进斑马鱼品系,并将这些品系以活体或冻存精子的形式进行合理的保存。目前CZRC已经和ZIRC建立正式合作,有计划地从ZIRC批量引进斑马鱼品系。2013年初,斑马鱼1号染色体全基因敲除计划(简称“ZKO计划”)正式启动,由中国科学院水生生物研究所、北京大学和清华大学等单位的38家实验室共同参与,历时近3年,获得了694个基因型明确的突变品系,目前这些品系均已在CZRC妥善保藏并对国内外学术界公开(http://www.zfish. cn/TargetList.aspx)。与此同时,CZRC利用TALEN和CRISPR/Cas9等基因组编辑技术和Tol2介导的转基因技术自主研制了超过200个基因敲除品系和转基因品系,有效地丰富了CZRC的品系资源库。
中心的斑马鱼品系资源使用活体养殖和冻存精子的形式进行维持和长期保藏。目前CZRC养殖的各类常用的活体斑马鱼品系有140余个,有力地保障了同行研究人员能够及时获取到宝贵的斑马鱼品系直接开展实验研究。为防止珍贵品系资源的丢失,CZRC的所有品系均以冻存精子的形式进行保藏。精子低温保藏技术是维持鱼类重要资源的一种长期、经济的方法,该技术的建立可以有效地节省大量的人力和物力成本,并且使鱼类重要品系的使用和维持更加灵活持久[10, 11]。经过5年时间的发展,CZRC建立了一个高品质的斑马鱼精子库。在经典的Harvey斑马鱼精子低温保藏术基础上[12],CZRC进一步优化了从精子收集到复苏操作的流程。到目前为止,CZRC的斑马鱼精子冷冻库已存储超过10 000管精子样本,冻存精子的平均复苏受精率为56.2% (图3B),居世界领先水平。总之,CZRC不仅有效地实现了斑马鱼品系资源在斑马鱼研究团体中的共享,同时精子冻存库的成功建立也为珍贵品系资源提供了一个有力的安全保障。
2.2 资源服务
历时5年,CZRC从2013年的资源总量不足100个增加到目前的1200多个品系资源。在建立之初,CZRC保藏的资源有限,2013年仅向国内研究者提供800多人次的斑马鱼品系资源服务。随着中心资源的丰富与多样化,在斑马鱼品系资源服务方面有了质和量的飞跃。2016年,斑马鱼品系资源服务次数提升至3000多人次,涉及斑马鱼品系、细胞、质粒、抗体、草履虫等多种资源(图3A)。CZRC不仅服务于国内的研究机构,也为美、法、德、日等海外国家和地区的实验室提供了斑马鱼资源服务。2015年3月,美国匹斯堡大学向CZRC一次订购了4种斑马鱼品系:CZ24 (ihb25)、CZ93 (ihb31)、CZ102 (gd2Gt)、CZ104 (ihb50)。CZRC克服重重困难,办理检验检疫、海关、外贸等多达19项审核材料和相关手续,最终将4个品系安全送抵匹兹堡大学,接收实验室已利用获得品系开展研究并发表相关论 文[13]。CZRC的服务获得了国内外研究同行的高度认可,截至2017年底,使用本中心提供品系资源所发表的相关文献累计超过100篇。CZRC的快速发展引起国际同行的关注,目前CZRC与ZIRC和欧洲斑马鱼资源中心(European Zebrafish Resource Center, EZRC)一同被并称为国际斑马鱼学界的三大资源库[14]。三大资源库均以提供斑马鱼品系资源为核心任务。经过近20的发展,ZIRC在线公布的品系信息达到38 738条,但其中26 259个突变品系是由英国Sanger研究所主持的斑马鱼突变筛选计划(Zebrafish Mutation Project, ZMP)提供[15]。如果计入Shawn和Lin实验室所提供的 逆转录病毒插入品系[16],则在ZIRC保藏的所有突变品系中,有 36 401 个品系的基因型均未被复核(unverified genotype,据ZIRC官方网站声明),这对研究人员索取和利用这些品系造成一定困难。EZRC在线列出22 346个品系,但其中也有19 980个品系是来源于ZMP计划。CZRC成立相对较晚,目前仅在线公布了1241个品系,但所有公布品系的基因型均经过确认。更值得一提的是,为了让研究人员更加方便快捷地获取品系用于研究,CZRC将140多个索取相对较频繁的品系以活体的形式进行养殖维护(表1)。虽然CZRC已经与Sanger研究所签订了资源引进协议,但是该研究所已将ZMP计划产生的所有突变品系冻存精子拷贝交由ZIRC和EZRC保藏,因此我国迫切需要在前期开展ZKO计划的基础之上,启动新的斑马鱼基因组定向突变和表型分析计划,以建立具有我国自主知识产权的斑马鱼资源品系库。
Table 1
表1
表1 ZIRC、EZRC、CZRC等全球三大斑马鱼资源库比较
Table 1
| 资源 中心 | 品系数目(未复核基因型品系/ZMP计划品系) | 活鱼 品系 | 固定 人员 | 主要经费来源渠道 | 网址及联系方式 | 提供的服务项目 |
|---|---|---|---|---|---|---|
| ZIRC | 38 738 (36 401/26 191) | 27 | 18 | 美国国立卫生研究院(稳定支持),俄勒冈大学,部分服务收入 | Web: http://zebrafish.org Email: zirc@zebrafish.org Phone: +1-541-346-6028 | 5种资源服务(Fish, ESTs/cDNAs, Antibodies, The zebrafish book, Paramecia) |
| EZRC | 22 346 (20 043/19 980) | 12 | 11 | 德国亥姆霍兹国家研究中心联合会(稳定支持),卡尔斯鲁厄理工学院 | Web: http://www.ezrc.kit.edu/ Email: EZRC-Requests@itg.kit.edu Tel.: +49-721-608-22716 | 2种资源服务(Fish, Plasmids) 3种技术服务(Sequencing and bioinformatics, Screening service, Meetings and Courses) |
| CZRC | 1241 (0/0) | 144 | 7 | 科技部国家重大科学研究计划(竞争性经费,已结题),中科院重点部署项目(竞争性经费,已结题),中科院科技服务网络计划补助经费 | Web: http://www.zfish.cn/ Email: zebrafish@ihb.ac.cn Phone: +86-27-6878-0570 | 6种资源服务(Fish, Plasmids, ESTs/cDNAs, Antibodies, Cell lines, Paramecia) 4种技术服务(Transgenic Service, Knock out Service, Zebrafish and health, Training Courses) |
新窗口打开|下载CSV
3 技术开发与服务
CZRC在确保高品质的品系资源服务的同时,积极构建斑马鱼品系创制相关技术平台,为斑马鱼研究科研人员提供及时有效的技术支撑。目前中心已经成功地建立了Tol2介导的转基因技术平台,以及TALEN 和CRISPR/Cas9介导的基因组编辑技术平台。3.1 转基因技术平台及服务
转基因技术的核心是将人工分离和修饰过的基因导入到生物体基因组中,由于导入基因的表达,引起生物体的性状产生可遗传的修饰[17, 18]。在构建转基因斑马鱼过程中,常用的导入外源基因的方式是通过显微注射技术,在显微镜下将外源基因通过注射导入斑马鱼受精卵中,外源基因可整合到基因组中,通过基因组筛选获得稳定遗传外源基因的品系[19]。转基因斑马鱼模型,尤其是特异组织细胞表达荧光蛋白的品系,被广泛应用于遗传学、发育生物学、医学、环境毒理学和水产育种学等研究领 域[20]。Tol2转座子系统是被广为使用的斑马鱼转基因工具。为进一步提高转基因表达效率,CZRC建立了高效、特异的斑马鱼原始生殖细胞转基因操作平台,可在胚胎发育期快速高效地筛选出生殖细胞整合有外源基因的P0代斑马鱼,大大提高了外源基因在斑马鱼中的表达效率和生殖传递效率[21,22]。CZRC不仅向斑马鱼研究人员提供个性化的转基因斑马鱼定制服务,还利用这一平台自主创制特异表达的工具类转基因品系,为斑马鱼研究提供了宝贵的研究材料。例如,CZRC利用转基因技术自主构建ihb20Tg品系,对外服务已达210人次。3.2 基因组编辑技术平台及服务
基因组编辑技术是人为地使靶向DNA序列发生碱基对的插入、缺失或替换,从而改变目标基因的结构和功能[18, 23],该技术是反向遗传学研究的重要技术手段。2011年,TALEN 技术以较为精准的打靶定位、构建简单易行和脱靶效应低等优势迅速取代锌指核酸酶技术,被广泛应用于包括斑马鱼等多个物种的基因编辑[24]。2012年出现的CRISPR/ Cas9技术[25,26]为基因编辑带来革命性的影响,效率极高、可操作性极强,迅速成为多数物种中构建基因敲除品系的主导技术。CZRC建立了基于TALEN技术的基因敲除平台和基于CRISPR/Cas9技术的基因敲除平台,均可高效特异地进行斑马鱼基因的敲除[27]。利用这个技术平台,中心不仅参与ZKO计 划,独立完成针对1号染色体基因的88个敲除品系构建,还根据学术界的需求自主构建了168个基因敲除品系,为学术界提供更多的研究工具。CZRC还建立了基于CRISPR/Cas9技术的斑马鱼原始生殖细胞基因敲除平台,该平台应用的转基因品系可在PGC中高效、特异表达斑马鱼偏好密码子Cas9,相比常规的Cas9敲除,可更加高效地筛选获得特定基因的敲除子代,为研制斑马鱼工具品系提供更加高效的技术支撑。目前,中心提供CRISPR/Cas9技术介导的斑马鱼基因敲除技术服务,并承诺在签订协议后140个工作日内提供2个具有不同移码框的突变品系。4 健康养殖、咨询和学术服务
4.1 斑马鱼养殖和疾病咨询服务
斑马鱼的健康养殖是开展一切与斑马鱼相关研究工作的基础。斑马鱼的生长、发育以及繁殖离不开良好的养殖环境。随着越来越多的实验室开展斑马鱼相关研究,科学的斑马鱼养殖技术和良好的鱼房管理制度显得尤为重要。CZRC从建立初始即努力为领域内提供斑马鱼养殖健康等咨询服务,介绍科学的养殖参数以及养殖要点。斑马鱼养殖环境重要参数主要有温度、水质参数(包括pH、硬度、氨氮水平、溶氧、盐度和电导率)、光周期和光照强度等,定期监控这些参数对维持斑马鱼的健康养殖至关重要[28]。CZRC希望通过这种咨询服务可以在一定程度上提高鱼房管理人员的科学养殖技术水平,以加强国内斑马鱼鱼房的健康发展。目前,越来越多的实验室利用循环水养殖系统建立了斑马鱼鱼房。鱼病问题一直困扰各大斑马鱼鱼房,不管是具明显可见病征或无明显可见病征的鱼类疾病都将危害实验用鱼的健康,影响实验研究结果,并经水体传播或直接接触导致鱼病在实验室之间相互传播。斑马鱼属于鲤科鱼类,故可感染鲤科鱼类动物的病原体均有可能感染斑马鱼[29]。斑马鱼的鱼病种类很多,根据发病机制,可分为两类:一类是非感染性疾病,一类是感染性疾病。两类疾病都可对斑马鱼的健康产生严重的危害,其中以感染性疾病造成的危害较为严重,常常可形成大规模的爆发式感染,严重影响斑马鱼的健康和实验结果的准确性。
在过去的3年中,CZRC工作人员考察了中国的20家主要的斑马鱼鱼房设施,发现在大多数设施中普遍存在着一些常见病症,例如竖鳞病、腹水、白点病、微孢子虫病、气泡病、肿瘤以及甲状腺肿等。多数鱼房存在着管理及实验操作上的问题,例如没有日常设施维护、缺乏水质监测系统、缺乏检疫程序、没有卫生措施和健康监测等,这些措施是维持良好养殖环境的必要条件,直接关系到鱼类健康和研究结果的准确性[30, 31]。因此,CZRC在向斑马鱼研究人员提供鱼病健康咨询的过程中,积极推广CZRC所实施的健康监测方案,包括环境和水质参数监测、定期设备维护、斑马鱼胚胎进口检疫 程序、消毒程序、卫生措施、卫生监督和疾病预防等[32, 33]。健康监控程序的有效施行可以防止新的斑马鱼病原体的引入,最大限度地减少已经存在的设施中的病原体,从而促进实验动物斑马鱼的健康和提高实验结果的有效性和可重复性。
4.2 斑马鱼相关学术服务
4.2.1 斑马鱼技术培训会议斑马鱼研究领域的发展,离不开优秀的青年科技人才队伍的发展壮大,从2014年开始,CZRC每年举办1~2届与斑马鱼研究相关的技术培训,到目前为止已经成功举办了八期全国培训会议。培训会议的对象主要针对中国斑马鱼研究领域的青年科研人员,培训专题包括斑马鱼鱼房建设和健康养殖、RNA原位杂交、显微注射、精子冻存和基因组编辑技术等斑马鱼实验技术。培训班采用理论课程与实验操作相结合的授课方式,学员在学习相关技术理论的同时,可以通过实际操作巩固所学技术要点。值得一提的是,在CZRC举办的斑马鱼基因组编辑技术培训会议中,学员在掌握该技术的同时,还可以对自己感兴趣的基因进行现场敲除,获得并带回敲除阳性的F0代胚胎。为了更好地掌握斑马鱼青年人才的切实需求和提高CZRC技术培训的质量,CZRC在每届培训班结束时进行匿名问卷评估,历届问卷评估的评分均达到9.0分以上(满分为10分)。在今后的培训会议中,CZRC要切实回应斑马鱼科研和产业领域的发展需求,进一步丰富课程内容,提高授课质量,提升理论和操作相结合的授课模式,以期更好地培养未来的斑马鱼科研人才。
4.2.2 全国斑马鱼PI大会
2011年,在广州举办的第二届全国斑马鱼研讨会上,包括孟安明院士在内的全国斑马鱼研究学术集体商定,今后的全国性斑马鱼会议采取“PI大会”和“研究大会”的形式交替隔年举行,并决定自2012年起固定在CZRC的依托单位水生所召开“全国斑马鱼PI大会”。至今为止,已成功举办了3届全国斑马鱼PI会议。在历届会议中,国内斑马鱼研究的科研带头人济济一堂,展示最新的斑马鱼相关的科研进展,寻求新的科研机遇。全国斑马鱼PI大会代表国内斑马鱼研究领域最高水平的学术会议,会议的主题主要有早期发育、信号通路、器官发育、疾病模型和环境健康等方面。目前我国有超过300家的实验室利用斑马鱼开展相关科学研究,通过全国斑马鱼PI大会平台,研究者可以更加全面地了解到斑马鱼研究相关的最新进展,获得许多宝贵的科研经验、知识以及启示,促进斑马鱼研究获得更高水平的科研产出。
4.2.3 中国动物学会斑马鱼分会成立
2016年9月,在武汉举行的第三届“全国斑马鱼PI会议”上,中国动物学会斑马鱼分会(简称中国斑马鱼学会,英文名China Zebrafish Society)正式成立。学会的挂靠单位为CZRC的依托单位水生所,秘书处设在CZRC,学会的名誉理事长由朱作言院士和孟安明院士担任,理事长由中国科学院动物研究所刘峰研究员担任,秘书长由CZRC主任孙永华研究员担任,常务委员会由17名成员构成,委员会包含54名成员。斑马鱼分会将遵守中国动物学会章程,根据斑马鱼的学科特点和国际发展趋势,开展相关工作。该学会的成立标志着中国的斑马鱼研究步入了新的发展阶段。
5 国际合作
鉴于斑马鱼作为模式动物在科研领域的重要作用和独特地位,在全球范围内,除CZRC外,美国、日本、澳大利亚、欧洲和中国台湾等国家和地区均设立有斑马鱼资源中心。目前全球最大的斑马鱼资源中心是ZIRC,位于美国俄勒冈州。ZIRC成立于1999年,现在保藏有全球斑马鱼研究者提供的将近3万多个斑马鱼品系,包括野生型、基因突变和转基因品系等。ZIRC还提供斑马鱼cDNA、抗体资源,以及养殖和疾病等方面的咨询服务。继ZIRC之后,欧洲、澳洲、日本、中国台湾等国家和地区都相继成立了提供斑马鱼资源保藏和服务的资源库。CZRC非常重视和ZIRC以及其他同类型斑马鱼资源库建立良好的合作关系。国际斑马鱼资源中心主任Monte Westerfield教授两次到访国家斑马鱼资源中心,CZRC也两次派工作人员访问国际斑马鱼资源中心,讨论有关资源交流和合作的细节。CZRC和ZIRC已签订资源交流协议;同时CZRC也与国际斑马鱼信息中心(Zebrafish Information Network, ZFIN)紧密合作,所有公布于CZRC的斑马鱼品系信息都会及时在ZFIN网站进行更新。这些合作将有助于提升我国斑马鱼研究和资源的国际展示度,促进我国斑马鱼研究的快速健康发展。
6 结语与展望
随着中国斑马鱼研究群体的迅速壮大,对各类综合信息和鱼房科学管理的需求更加迫切。CZRC正着手为国内斑马鱼科研工作者打造中国斑马鱼信息中心(Chinese Zebrafish Information Network, CZIN, 网址:http://zfin.cn),CZIN将不仅为中国斑马鱼群体提供学术展示与交流的平台,同时还将开发品系管理的云平台,为各实验室管理日益增加的品系资源提供更加高效可靠的数字平台。品系共享和信息化服务将有助于建立CZRC以及国内外斑马鱼实验室之间的合作及协调关系,最大限度地满足斑马鱼研究****对斑马鱼相关资源和信息的需求。目前,CZIN的学术展示和交流平台已经正式上线开放。总之,CZRC将遵循中心章程[34],以斑马鱼研究资源的收集、创制、整理、保藏和分享为主要任务,以服务于全国斑马鱼研究****为宗旨,不忘初心,不断进取,整合资源,促进合作,为我国斑马鱼研究事业的长期健康发展奠定基础。
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The zebrafish (Danio rerio) provides an excellent model for studying vertebrate development and human disease because of its ex utero, optically transparent embryogenesis and amenability to in vivo manipulation. The rapid embryonic developmental cycle, large clutch sizes and ease of maintenance at large numbers also add to the appeal of this species. Considerable genomic data has recently become publicly available that is aiding the construction of zebrafish microarrays, thus permitting global gene expression analysis. The zebrafish is also suitable for chemical genomics, in part as a result of the permeability of its embryos to small molecules and consequent avoidance of external confounding maternal effects. Finally, there is increasing characterization and analysis of zebrafish models of human disease. Thus, the zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effect of small molecules as well as for dissecting biological pathways.
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斑马鱼作为重要的脊椎动物模式系统之一, 由于其多方面的优势, 在生命科学研究领域发挥着越来越重要的作用。目前, 斑马鱼已被广泛地用于发育生物学、分子生物学、细胞生物学、遗传学、神经生物学、肿瘤学、免疫学、海洋生物学、药物学、毒理与环保等诸多方面的研究, 一些重要的成果不断涌现, 为现代生命科学的发展做出了重要贡献。我国20世纪90年代后期引入斑马鱼模式系统, 此后研究队伍扩大很快, 有影响的研究成果不断涌现, 促进了多个学科的发展。文章重点综述了我国内地与香港地区在斑马鱼研究方面的发展历程以及所取得的代表性成果, 以期促进该模式系统更广泛地用于开展高水平研究。
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斑马鱼作为重要的脊椎动物模式系统之一, 由于其多方面的优势, 在生命科学研究领域发挥着越来越重要的作用。目前, 斑马鱼已被广泛地用于发育生物学、分子生物学、细胞生物学、遗传学、神经生物学、肿瘤学、免疫学、海洋生物学、药物学、毒理与环保等诸多方面的研究, 一些重要的成果不断涌现, 为现代生命科学的发展做出了重要贡献。我国20世纪90年代后期引入斑马鱼模式系统, 此后研究队伍扩大很快, 有影响的研究成果不断涌现, 促进了多个学科的发展。文章重点综述了我国内地与香港地区在斑马鱼研究方面的发展历程以及所取得的代表性成果, 以期促进该模式系统更广泛地用于开展高水平研究。
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Fishes,the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics.Nuclear transplantation in fish has been thoroughly studied in China since 1960s.Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults.Most importantly,nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish.This was the first case of cloned fish with somatic cells.Based on the technique of microinjection,recombinant MThGH gene has been transferred into fish eggs and the firsh batch of transgenic fish were produced in 1984.The behavior of foreign gene was characterized and the onsed of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis.This eventually led to the transgenic mosaicism.The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults.The transgenic common carp were more efficient in utilizing dietary protein than the controls.An “all-fish” gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (grGH) coding sequence.The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait.Combination of techniques of fish cell culture,gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21^st century.
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斑马鱼是一种新兴的脊椎模式动物。在过去的30年中,斑马鱼已被广泛应用于生命科学、健康科学、环境农业等诸多科研领域。为了满足不同的科研需要,研究人员开发和利用各种技术创建了大量的斑马鱼基因突变和转基因品系,这些品系已成为开展相关科学研究的宝贵资源。为了更好地保藏和利用这些资源,在全球范围内建设有多个规模不一的斑马鱼资源库。2012年,我国的国家斑马鱼资源中心(http://zfish.cn)在中国科学院水生生物研究所正式成立。本文将重点介绍全球斑马鱼资源的开发和保藏情况,以及我国国家斑马鱼资源中心的最新建设进展。
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斑马鱼是一种新兴的脊椎模式动物。在过去的30年中,斑马鱼已被广泛应用于生命科学、健康科学、环境农业等诸多科研领域。为了满足不同的科研需要,研究人员开发和利用各种技术创建了大量的斑马鱼基因突变和转基因品系,这些品系已成为开展相关科学研究的宝贵资源。为了更好地保藏和利用这些资源,在全球范围内建设有多个规模不一的斑马鱼资源库。2012年,我国的国家斑马鱼资源中心(http://zfish.cn)在中国科学院水生生物研究所正式成立。本文将重点介绍全球斑马鱼资源的开发和保藏情况,以及我国国家斑马鱼资源中心的最新建设进展。
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Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-alpha, Hif2-alpha and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors.
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In zebrafish, maternally produced vasa ( vas) transcripts become targeted to the cleavage planes of early embryos and subsequently incorporated into the primordial germ cells (PGCs). Zygotic vas transcription occurs from the onset of gastrulation. Here, we report on the characterisation of the zebrafish vas locus. The gene consists of 27 exons, spans about 25 kb, and contains two CpG-rich regions. We have used vas regulatory regions to establish transgenic zebrafish lines expressing enhanced green fluorescent protein (EGFP) in their PGCs. Maternally encoded vas::EGFP transcripts and VAS::EGFP protein segregate with the PGCs during embryogenesis. We find that the maternally deposited vas::EGFP transcripts are stable during embryogensis at least up to 50 h of development. Vas::EGFP transcripts could not be detected in embryos that inherit the transgene from males, most likely due to the lack of one or more regulatory elements required for early zygotic expression. We show that vas::EGFP transcripts become enriched to the cleavage planes in early embryos, a finding that supported an RNA localisation signal localised within the vas region of these transcripts.
URLPMID:17644987 [本文引用: 1]
Abstract Initial success in sperm cryopreservation came at about the same time for aquatic species and livestock. However, in the 50-plus years since then cryopreserved sperm of livestock has grown into a billion-dollar global industry, while despite work in some 200 species with well over 200 published reports, cryopreservation of aquatic species sperm remains essentially a research activity with little commercial application. Most research has focused on large-bodied culture and sport fishes, such as salmonids, carps, and catfishes, and mollusks such as commercially important oyster and abalone species. However, only a handful of studies have addressed sperm cryopreservation in small fishes, such as zebrafish, and in endangered species. Overall, this work has yielded techniques that are being applied with varying levels of success around the world. Barriers to expanded application include a diverse and widely distributed literature base, technical problems, small sperm volumes, variable results, a general lack of access to the technology, and most importantly, the lack of standardization in practices and reporting. The benefits of cryopreservation include at least five levels of improvements for existing industries and for creation of new industries. First, cryopreservation can be used to improve existing hatchery operations by providing sperm on demand and simplifying the timing of induced spawning. Second, frozen sperm can enhance efficient use of facilities and create new opportunities in the hatchery by eliminating the need to maintain live males, potentially freeing resources for use with females and larvae. Third, valuable genetic lineages such as endangered species, research models, or improved farmed strains can be protected by storage of frozen sperm. Fourth, cryopreservation opens the door for rapid genetic improvement. Frozen sperm can be used in breeding programs to create improved lines and shape the genetic resources available for aquaculture. Finally, cryopreserved sperm of aquatic species will at some point become an entirely new industry itself. A successful industry will require integrated practices for sample collection, refrigerated storage, freezing, thawing, rules for use and disposal, transfer agreements, and database development. Indeed the development of this new industry is currently constrained by factors including the technical requirements for scaling-up to commercial operations during the transition from research, and the absence of uniform quality control practices, industry standards, marketing and price structures, and appropriate biosecurity safeguards.
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We describe a method for cryopreservation of milt from individual using methanol and powdered milk as cryoprotectants. Motility was positively correlated with hatching, which averaged 5168±6835.6% in a typical experiment. Variablity in motility and hatching was not correlated with sperm volume or age of the fish, and is believed to be due to differences in sperm quality between individuals, as well as technical constraints imposed by the short duration of motility in thawed spermatozoa.
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Bromodomain and extraterminal (BET) proteins recruit key components of basic transcriptional machinery to promote gene expression. Aberrant expression and mutations in BET genes have been identified in many malignancies. Small molecule inhibitors of BET proteins like JQ1 have shown efficacy in preclinical cancer models including affecting growth of hepatocellular carcinoma. BET proteins also... [Show full abstract]
URLPMID:26097180 [本文引用: 1]
Summary <p>The Zebrafish Model Organism Database (ZFIN;
URLPMID:3743023328411408000549669101 [本文引用: 1]
Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches(1-3) and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes(3,4), this number falls considerably short of the more than 22,000 mouse protein-coding genes(5). Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning(6), insertional mutagenesis(7-9), antisense morpholino oligonucleotides(10), targeted re-sequencing(11-13), and zinc finger and TAL endonucleases(14-17) have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes(18). Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence(18,19), high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.
URLPMID:23382537 [本文引用: 1]
With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived Firs predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized similar to 0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.
URLPMID:25394772 [本文引用: 1]
The use of transgenics in fish is a relatively recent development for advancing understanding of genetic mechanisms and developmental processes, improving aquaculture, and for pharmaceutical discovery. Transgenic fish have also been applied in ecotoxicology where they have the potential to provide more advanced and integrated systems for assessing health impacts of chemicals. The zebrafish (Daniorerio) is the most popular fish for transgenic models, for reasons including their high fecundity, transparency of their embryos, rapid organogenesis and availability of extensive genetic resources. The most commonly used technique for producing transgenic zebrafish is via microinjection of transgenes into fertilized eggs. Transposon and meganuclease have become the most reliable methods for insertion of the genetic construct in the production of stable transgenic fish lines. The GAL4-UAS system, where GAL4 is placed under the control of a desired promoter and UAS is fused with a fluorescent marker, has greatly enhanced model development for studies in ecotoxicology. Transgenic fish have been developed to study for the effects of heavy metal toxicity (via heat-shock protein genes), oxidative stress (via an electrophile-responsive element), for various organic chemicals acting through the aryl hydrocarbon receptor, thyroid and glucocorticoid response pathways, and estrogenicity. These models vary in their sensitivity with only very few able to detect responses for environmentally relevant exposures. Nevertheless, the potential of these systems for analyses of chemical effects in real time and across multiple targets in intact organisms is considerable. Here we illustrate the techniques used for generating transgenic zebrafish and assess progress in the development and application of transgenic fish (principally zebrafish) for studies in environmental toxicology. We further provide a viewpoint on future development opportunities.
URLPMID:26654901 [本文引用: 2]
Abstract. Zebrafish is a powerful model for the study of vertebrate development, being amenable to a wide range of genetic and other manipulations to probe the
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URLPMID:16127723 [本文引用: 1]
The zebrafish, originally an object of study as an inexpensive and prolific vertebrate embryological model with a plethora of genetic tricks, has over the past decade moved to large-scale chemical mutagenesis and recently came of age as a high throughput transgenic model with a sequenced genome nearing completion. Insertional mutagenesis, gene trapping and enhancer detection are all contributing to the increasing speed with which research in this biomedical model is progressing. We review here some of the recent developments in the emerging field of zebrafish developmental genomics and transgenesis. Developmental Dynamics 234:255-268, 2005. 2005 Wiley-Liss, Inc.
URLPMID:23535913 [本文引用: 1]
In zebrafish and other vertebrates, primordial germ cells (PGCs) are a population of embryonic cells that give rise to sperm and eggs in adults. Any type of genetically manipulated lines have to be originated from the germ cells of the manipulated founders, thus it is of great importance to establish an effective technology for highly specific PGC-targeted gene manipulation in vertebrates. In the present study, we used the Cre/loxP recombinase system and Gal4/UAS transcription system for induction and regulation of mRFP (monomer red fluorescent protein) gene expression to achieve highly efficient PGC-targeted gene expression in zebrafish. First, we established two transgenic activator lines, Tg(kop:cre) and Tg(kop:KalTA4), to express the Cre recombinases and the Gal4 activator proteins in PGCs. Second, we generated two transgenic effector lines, Tg(kop:loxP-SV40-loxP-mRFP) and Tg(UAS:mRFP), which intrinsically showed transcriptional silence of mRFP. When Tg(kop:cre) females were crossed with Tg(kop:loxP-SV40-loxP-mRFP) males, the loxP flanked SV40 transcriptional stop sequence was 100 % removed from the germ cells of the transgenic hybrids. This led to massive production of PGC-specific mRFP transgenic line, Tg(kop:loxP-mRFP), from an mRFP silent transgenic line, Tg(kop:loxP-SV40-loxP-mRFP). When Tg(kop:KalTA4) females were crossed with Tg(UAS:mRFP) males, the hybrid embryos showed PGC specifically expressed mRFP from shield stage till 25 days post-fertilization (pf), indicating the high sensitivity, high efficiency, and long-lasting effect of the Gal4/UAS system. Real-time PCR analysis showed that the transcriptional amplification efficiency of the Gal4/UAS system in PGCs can be about 300 times higher than in 1-day-pf embryos. More importantly, when the UAS:mRFP-nos1 construct was directly injected into the Tg(kop:KalTA4) embryos, it was possible to specifically label the PGCs with high sensitivity, efficiency, and persistence. Therefore, we have established two targeted gene expression platforms in zebrafish PGCs, which allows us to further manipulate the PGCs of zebrafish at different levels.
URLMagsci [本文引用: 1]
<p>利用斑马鱼作为体内模型, 研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells, PGCs)中同源重组(Homologous recombination, HR)的效率。首先, 将<em>UAS:mRFP-nos</em>1载体显微注射到<em>Tg </em>(<em>kop:KalTA</em>4) 转基因胚胎中标记转基因PGCs, 结果表明筛选PGCs特异表达mRFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系, 并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平, 而且在PGCs水平都能够显著提高HR的效率。研究表明<em>Tg </em>(<em>kop:KalTA</em>4) 转基因品系是开展HR介导的基因打靶的一个有效平台。</p>
URLMagsci [本文引用: 1]
<p>利用斑马鱼作为体内模型, 研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells, PGCs)中同源重组(Homologous recombination, HR)的效率。首先, 将<em>UAS:mRFP-nos</em>1载体显微注射到<em>Tg </em>(<em>kop:KalTA</em>4) 转基因胚胎中标记转基因PGCs, 结果表明筛选PGCs特异表达mRFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系, 并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平, 而且在PGCs水平都能够显著提高HR的效率。研究表明<em>Tg </em>(<em>kop:KalTA</em>4) 转基因品系是开展HR介导的基因打靶的一个有效平台。</p>
URLPMID:19444792 [本文引用: 1]
The analysis of mutant organisms and cell lines is important in determining the function of specific proteins. Recent technological advances in gene targeting by homologous recombination in mammalian systems enable the production of mutants in any desired gene, and can be used to produce mutant strains and mutant cell lines. The /system and such as and its recognition sequence, loxP, allow spatial and temporal control of knockouts. This unit discusses crucial issues for homologous recombination experiments, including requirements for the source of DNA, criteria for the targeting constructs, methods of enrichment for homologous recombinants, (positive and negative selection, and the use of endogenous promoters), and the types of mutations that can be created.
URLPMID:21822242 [本文引用: 1]
Nat Biotechnol. 2011 Aug 5;29(8):699-700. doi: 10.1038/nbt.1939. Comment; Letter; Research Support, Non-U.S. Gov't
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尽管早在1891年就有因嗜水气单胞菌(<i>Aeromonas hydrophila</i>)感染引致蛙“红腿病”的报道,但由于该菌在自然界尤其是水中广泛分布,一般为正常共栖菌,因此长期以来对其致病性并未重视。近年来在我国南方各省淡水养殖鱼类流行暴发性传染病,有报道系为该菌所致败血症。此外在其它水生经济动物、哺乳动物亦有类似发现。人类因之而发生胃肠炎及伤口感染的病例也日渐增多。嗜水气单胞菌致病问题已跃然成为当代公共卫生瞩目的对象，为人们提出了人一畜一鱼共患病的研究新课题。本文介绍该菌的分类地位、病原特性、与鱼有关的流行病学及病原分离与鉴定方面的研究进展。
Magsci [本文引用: 1]
尽管早在1891年就有因嗜水气单胞菌(<i>Aeromonas hydrophila</i>)感染引致蛙“红腿病”的报道,但由于该菌在自然界尤其是水中广泛分布,一般为正常共栖菌,因此长期以来对其致病性并未重视。近年来在我国南方各省淡水养殖鱼类流行暴发性传染病,有报道系为该菌所致败血症。此外在其它水生经济动物、哺乳动物亦有类似发现。人类因之而发生胃肠炎及伤口感染的病例也日渐增多。嗜水气单胞菌致病问题已跃然成为当代公共卫生瞩目的对象，为人们提出了人一畜一鱼共患病的研究新课题。本文介绍该菌的分类地位、病原特性、与鱼有关的流行病学及病原分离与鉴定方面的研究进展。
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