斑马鱼资源的开发保藏与国家斑马鱼资源中心

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。     

斑马鱼基因工程的研究进展

对国内外斑马鱼基因工程研究进展。包括最近获得的大批转基因斑马鱼品系、靶向筛选的转基因斑马鱼、斑马鱼转基因技术的发展和斑马鱼基因工程的热点问题与应用前景进行了综述。指出我国已建立日趋成熟的斑马鱼转基因技术、细胞核移植技术和染色体组操作技术,具有斑马鱼细胞遗传学和胚胎学基础,我国有望在不久的将来获得定向整合的基因工程斑马鱼,并推动生物技术应用研究领域的进步和发展。     

斑马鱼超弱光特性的初步研究

本文报道有关鱼类(斑马鱼)超弱发光的新资料:光谱分布、发光动力学曲线及有关参数与pH,O_2,剥鳞和化学物质对超弱发光的影响,以及鱼体表面积和鱼类检测时的精度和准度。     

斑马鱼心脏再生的研究

人类心脏损伤后不可再生,使得心脏疾病成为威胁人类生命的主要疾病之一。斑马鱼等其他非哺乳动物类脊椎动物的心脏在受损伤后可以再生,再加上其遗传操作已十分成熟,使其成为研究脊椎动物心脏再生的一个极好模型。本文总结了国内外关于斑马鱼心脏再生的最新进展、并分析了心脏再生特异的分子机制以及涉及再生现象的保守机制,进一步提出了这些研究对于人类心脏疾病的治疗的可借鉴之处。     

斑马鱼研究走向生物医学

1972年美国俄勒冈大学George Streisinger教授开始研究斑马鱼(Danio rerio)至今, 斑马鱼以其独特的优点, 已经成为现代遗传学、发育生物学研究的重要模式动物。世界范围内斑马鱼研究群体的工作已奠定了较为完善的胚胎学、分子遗传学研究基础, 并且斑马鱼已被应用于开发人类重大疾病模型和药物筛选平台, 取得了许多有价值的研究成果。文章简述了斑马鱼成为模式动物的历史, 侧重介绍了业已建立的白血病、黑色素瘤、感染免疫疾病、神经疾病等斑马鱼模型, 以及利用斑马鱼进行小分子化合物/药物筛选和研发的现状。斑马鱼研究向生物医学方向的拓展, 必将为人类理解重大疾病发生机制、寻找疾病治疗方法, 为维护人类卫生、健康做出贡献。     

生长激素促进斑马鱼尾鳍的再生

目的建立生长激素过表达的转基因斑马鱼,研究生长激素在斑马鱼尾鳍再生过程中的作用。方法利用Gateway技术构建表达质粒"pDestTol2CG2; ubi:GH-polyA",在一细胞期显微注射表达质粒和转座酶mRNA后,通过荧光显微镜和qPCR技术筛选鉴定GH过表达的转基因斑马鱼。将斑马鱼分为对照组(野生型)和生长激素过表达组,尾鳍横切后,记录分析斑马鱼尾鳍再生过程。结果转基因斑马鱼中心脏被绿色荧光蛋白标记。荧光定量PCR检测结果显示GH表达水平显著高于对照组(P<0.05)。斑马鱼尾鳍横断后,生长激素过表达组的再生速度显著提高(P<0.05)。结论建立稳定生长激素过表达的转基因斑马鱼品系,过表达生长激素能够提高斑马鱼尾鳍再生速度。     

斑马鱼脾显微与超微结构的研究

应用光学显微镜和透射电镜对斑马鱼(Daniorerio)脾的显微与超微结构进行了观察。光镜结果表明,斑马鱼的脾主要由脾髓和网状组织两部分构成,脾髓分布于整个脾,分为白髓和红髓,但界限不明显,呈混合分布。红髓染色相对较浅,占脾实质的大部分区域,主要由密集的红细胞构成,可见脾窦结构,脾索结构不明显。白髓染色较深,主要由密集的淋巴细胞构成,淋巴细胞的胞核染色较深,呈深紫色。经Gordon-Sweet银染显示网状纤维后,可清晰观察到脾小结,从而可清晰区分红髓与白髓,脾小结与哺乳动物的类似,主要是由密集的淋巴细胞构成。酸性磷酸酶染色可见斑马鱼脾内有大量吞噬性细胞存在。电镜结果显示,红髓分布有不同类型的红细胞、血小板和浆细胞,白髓分布有淋巴细胞、巨噬细胞、单核细胞、嗜中性粒细胞、浆细胞和网状细胞,淋巴细胞胞质内含有多泡体,这可能与淋巴细胞发挥免疫功能有着密切联系。斑马鱼脾与大多数硬骨鱼相似,未观察到椭球这一特殊结构。     

中国斑马鱼研究发展历程及现状

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。     

斑马鱼:在生命科学中畅游

模式动物是生命科学研究的重要材料,为生命科学的发展做出了重要的贡献。自20世纪90年代初以来,斑马鱼因其多方面的优点已成为模式动物家族中重要的一员,受到越来越多的重视和利用。目前,斑马鱼被广泛地用于发育生物学、遗传学、肿瘤学、药物学、毒理与环保等方面的研究,不断涌现新的研究成果。     

斑马鱼干扰素γ的克隆表达研究

目的：通过人工诱导斑马鱼干扰素的产生,证实两种斑马鱼干扰素γ基因ifng1-1和ifng1-2的存在,并克隆这两种干扰素基因,构建高效表达载体并做原核表达。方法：通过conA药浴,诱导斑马鱼干扰素γ的表达,即刻提取组织总RNA,并通过RT-PCR方法扩增两种干扰素γ基因。将目的基因连入表达载体pET24a,构建重组载体pET24a-ifng1-1和pET24a-ifng1-2。将载体转化BL21,并用IPTG诱导融合蛋白的表达。产物经SDS-PAGE检测,分析蛋白表达情况。结果：成功克隆了两种斑马鱼干扰素γ基因,构建了pET24a-ifng1-1和pET24a-ifng1-2重组载体,并实现了原核融合表达。结论：两种干扰素γ基因都能够被conA所诱生,且具有相似特性,表明两种干扰素γ都不是假基因,都有可能在免疫系统中发挥作用,本实验为进一步研究两种干扰素γ的功能奠定了基础。     

Status, problems and prospects of stock enhancement in Taiwan

Stock enhancement started in Taiwan with the building and casting of artificial reefs in 1973. It was only in late 1987, however, that an integrated program on the operation and establishment of a stock enhancement system was developed. In addition to building artificial reefs and establishing resource protection zones to create fishing grounds, the current stock enhancement program in Taiwan aims at restocking broodstock and fry or seeds. So far, seven species of finfish, four species of mollusks, and six species of crustaceans have been restocked. A total of 5.8 million finfish fry, 5 million molluskan seeds, and 30 million crustacean larvae with some eel and prawn broodstocks have been released up to 1996. Although there are plans to establish sea-farming centers in Taiwan, the organization of the system has not been established completely. Furthermore, regulations on resource augmentation, protection and management of coastal fisheries and stock enhancement are absent or lacking. The success of stock enhancement depends on the pollution control that is in place in coastal areas and the fishermen‘s awareness of the importance of resource conservation and their involvement. The paper discusses the status,problems and prospects of stock enhancement in Taiwan, with some viewpoints in population genetics. It also refers to some experience in Japanese sea-farming. This revised version was published online in August 2006 with corrections to the Cover Date.     

Baculovirus-Mediated Gene Expression in Zebrafish

Abstract In an effort to misexpress genes in zebrafish, we tested the ability of baculovirus to infect and drive gene expression in embryos. By injecting virus into specific tissues and using appropriate promoters, both the location and time of gene expression could be controlled. Using a virus with 2 different promoters, LacZ and GFP could be expressed independently. The efficiency of expression appears to depend on the promoter used. As a test of this system, baculovirus was used to ectopically express ephrinB2a in the presomitic mesoderm. EphrinB2a is normally expressed in the posterior region of developing somites, and baculovirus-mediated misexpression caused abnormal somite boundary formation. Baculovirus can thus be used as a tool for gene misexpression experiments in the zebrafish, especially when localized misexpression is required late in development.     

斑马鱼在新药发现中的应用

在过去20年里,斑马鱼已成为一种重要的模式脊椎动物,在发育、遗传、免疫、肿瘤和毒理等诸多研究领域中被广泛应用。近年来,斑马鱼作为活体模型越来越多地应用于某些生物学过程的药物筛选。通过斑马鱼初步筛选,在药物研发初期可确定化合物的生物学活性、毒性以及副作用等。最近的研究还发现,斑马鱼不仅用于新药筛选,还可用于药物结构的优化。本文重点介绍斑马鱼在新药发现中的应用。     

RecA-Mediated,Targeted Mutagenesis in Zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into 1-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in 1 or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems. Current address (Ying Yang): First Hospital of Beijing University, Beijing 100034, P.R. China. Current address (Christopher D. Kaufman): Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse 10, D-13092 Berlin, Germany. Current address (Perry B. Hackett): Discovery Genomics, Inc. 614 McKinley Pl. NE Minneapolis, MN 55413     

Caspase-3 Induced Apoptosis in Transgenic Zebrafish

Zebrafish is an attractive model organism for studying apoptosis development because of its genetic accessibility. Here we describe the induction of clonally derived apoptosis in transgenic zebrafish expressing mouse caspase-3 (CASP3) under control of the zebrafish β-actin promoter (βp). Visualization of apoptotic cells, expressing a chimeric transgene encoding CASP3 fused to green fluorescent protein (GFP) gene, revealed that apoptosis arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into abdominal organs like testis, kidney. This transgenic model provides a platform for over-expression of caspase-3 induced extensive apoptosis in embryos and adult. An erratum to this article is available at .     

Parvalbumin Isoforms in Zebrafish

By using an analysis of existing genomic information it is concluded that in zebrafish nine genes encode parvalbumin (PV). These genes possess introns that differ in size and show nucleotide variability but they contain the same number of exons, and for each corresponding exon, the number of nucleotides therein are identical in all the paralogs. This rule also applies to the multiple PV genes of other species e.g. mammals. Each of these genes displays, however, characteristic 5′ and 3′ UTRs which appear highly conserved between closely related species (so that orthologs among these species can be readily identified) but which show larger numbers of mutations between species that are more distant in evolution. A tree is presented which suggests that the traditional classification of PVs as alpha or beta (based mainly on charge of the protein molecule) is not sustainable. Numbers 1–9 are assigned to the various isoforms to facilitate their identification in future studies. A bifurcation of isoforms into 1 and 4; 2 and 3; 6 and 7; 8 and 9 appears to have occurred simultaneously in more recent time, i.e. perhaps approximately 60 mys ago when primates and rodents branched.     

斑马鱼母源因子在胚胎发育中的作用

动物受精时,精子主要是将雄原核释放到卵子中,形成的合子中雌、雄原核融合为合子核,但受精卵基因组在前几次有丝分裂过程中不转录,合理的逻辑性推测是其早期发育完全依赖于卵质中储存的RNA和蛋白质,即母源因子.上世纪80年代对无脊椎动物的正向遗传研究发现,母源因子在卵子和胚胎极性的决定、早期胚胎的图式形成等方面发挥了决定性作用.过去10多年来,通过对斑马鱼和小鼠突变体的研究,也证明母源因子在脊椎动物胚胎早期发育中起着重要作用.本文主要综述斑马鱼母源因子在卵母细胞的极性、卵子的激活、早期细胞分裂、母源mRNA的清除、合子基因转录激活以及胚层的形成和分化、体轴的建立等方面的作用,相关知识对于研究人类生育障碍和先天性疾病的发生机制和诊治有借鉴意义.     

Zebrafish model of KRAS-initiated pancreatic endocrine tumor

Pancreatic cancer constitutes a genetic disease in which somatic mutations in the KRAS proto-oncogene are detected in 95% of cases. Activation of the KRAS proto-oncogene represents an initiating event in pancreatic tumorigenesis. Here, we established a zebrafish pancreatic neoplasia model that recapitulates human pancreatic tumors. Toward this end, we generated a stable CRE/Lox-based zebrafish model system to express oncogenic KRAS^G12D  in the elastase3I domain of the zebrafish pancreas. Lineage tracing experiments showed that early KRAS^G12D -responsive pancreatic progenitors contribute to endocrine in addition to exocrine cells. In this system, 10% and 40% of zebrafish developed pancreatic tumors by 6 and 12 months, respectively. The histological profiles of these experimental tumors bore a striking resemblance to those of pancreatic endocrine tumors. Immunohistochemical analysis including the endocrine cell-specific marker confirmed the pancreatic tumor region as a characteristic endocrine tumor. Taken together, our zebrafish model data revealed that pancreatic endocrine tumors originate from early KRAS^G12D -responsive pancreatic progenitor cells. These findings demonstrated that this zebrafish model may be suitable as an experimental and preclinical system to evaluate different strategies for targeting pancreatic endocrine tumors and ultimately improve the outcome for patients with pancreatic endocrine tumors.     

组蛋白精氨酸甲基化与斑马鱼早期发育

发育是由基因的特定时空表达模式来调控的,其表观遗传机制已越来越受到关注。组蛋白精氨酸甲基化是一种重要的翻译后修饰,由蛋白质精氨酸甲基化酶催化产生,对染色体的结构与功能具有重要调控作用。不同位点的精氨酸甲基化与其相邻位点的翻译后修饰具有复杂的对话机制,并可招募或阻碍相关效应分子的结合,进而导致转录激活或抑制。斑马鱼作为一种重要的发育生物学研究模式动物,已为蛋白质精氨酸甲基化酶在早期发育过程中的生理功能的研究提供了大量资料。该文对组蛋白精氨酸甲基化的产生、对话调控机制及其对斑马鱼早期发育调控功能的研究进行综述。