一种可高效转染的青鳉肌肉细胞系的建立与应用（英文）

在哺乳动物细胞系中转基因的效率相对较高而在鱼类细胞系中相对较低,为了获得鱼类高效转基因细胞系,我们从模式鱼类青鳉(Oryzias latipes)中分离和构建了肌肉细胞系OLM。该细胞系类似于纤维细胞,在28℃DMEM和10%的胚胎牛血清中培养了超过88代。通过染色体分析,它属于两倍体并含有48条染色体。在脂质体介导下,报告基因的转染效率可以达到40%,在测试的其他鱼类细胞中转染效率最高。此外还构建了稳定转染转基因或者非编码RNA的细胞系。OLM细胞对常见的病毒SVCV、GCRV、SGIV等不敏感。综上所述,研究在青鳉中建立了一种能高效转染的肌肉细胞系,它适用于鱼类瞬时和稳定的转基因研究。     

金鱼hAT家族转座子Tgf2的克隆及其结构

hAT家族转座子以果蝇hobo、玉米Ac和金鱼草(Ceratophyllum demersum L.)Tam3为代表,以"剪切-粘帖"方式进行DNA转座。1996年,日本学者首次在白化青鳉(Oryzias latipes)中发现具有天然活性的脊椎动物hAT家族转座子,即青鳉Tol2转座子,该转座子已在模式生物斑马鱼转基因、基因和启动子捕获方面进行了广泛应用。文章根据玉米Ac与青鳉Tol2转座子序列保守区设计一对引物,在19种不同鱼类物种或品系中进行PCR筛选,最后发现此类hAT家族转座子在我国不同品系金鱼中存在,命名为金鱼Tgf2转座子。金鱼Tgf2转座子全长4720bp,由4个阅读框组成,与青鳉Tol2转座子的相似度为97%。金鱼Tgf2与青鳉Tol2转座子在末端倒位重复和亚末端重复上存在一定差异,此外,金鱼Tgf2转座子的中间反向重复序列(1453bp到2091bp)可形成一种"十"字结构,明显有别于青鳉Tol2转座子形成的茎环结构,这些区域与转座活性密切相关。文章预示金鱼Tgf2转座子可能具有更高的天然转座活性,构建高效金鱼Tgf2转基因元件可供鱼类转基因和基因捕获研究。     

鱼类的胚胎干细胞

胚胎干细胞(ES)是未分化的细胞培养物，来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下，ES细胞能分化成各种细胞类型，包括生殖细胞。这样，ES细胞就提供了一个有效的纽带，将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前，ES细胞技术仅见之老鼠，因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类，人们已做了大量的尝试。我们以青鳉(Oryzias latipes)作为建立鱼类ES细胞技术的模式，通过建立并应用无滋养层细胞的培养条件，获得了来自中期囊胚的ES细胞系。青鳉的ES细胞和老鼠的ES细胞有很多共同特征，如二倍体核型、分化潜力和形成嵌合体。因此，在鱼类建立和应用ES细胞技术是可能的。青鳉ES细胞的培养条件已成功地应用到其它鱼类如斑马鱼甚至海水鱼。本文旨在以青鳉为模式，综述获得和应用模式鱼和经济鱼ES细胞的主要进展和前景。     

青鳉与食蚊鱼竞争排斥的生态形态学解释

生态形态学理论指出,形态相似的物种生态位相似,是导致种间竞争排斥的关键性因素。在鱼类入侵生态学研究中引入生态形态学理论,对于加深理解入侵种与土著种的种间相互关系有着重要意义。本文利用形态分析方法,对广东怀集燕都国家湿地公园入侵种食蚊鱼对土著种鳍斑青鳉和弓背青鳉的影响机制进行研究。结果表明: 弓背青鳉和鳍斑青鳉在研究区域常同域分布,但两者的空间分布格局差异显著。两者形态高度相似,符合生态形态学“形态相似者竞争排斥”理论。与群落其他鱼类相比,食蚊鱼与2种青鳉鱼类形态更为相似。聚类分析发现,食蚊鱼和青鳉鱼类聚合在同一分枝,且其空间生态位的重叠度极低。种群相对密度调查显示,食蚊鱼与青鳉鱼类种群数量呈显著的负相关关系。食蚊鱼的入侵是导致2种青鳉鱼类种群数量明显下降的关键原因。形态特征的相似性能够初步解释食蚊鱼与青鳉鱼类的竞争排斥关系,尚需从不同角度就食蚊鱼入侵对青鳉鱼类的作用机制进行深入探索。     

一种简便高效的人胚胎干细胞转染方法

目的 :利用Fugene 6基因转染试剂建立一种简便高效的人胚胎干细胞转染方法 ,并建立稳定表达增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)报告基因的人胚胎干细胞系 ,为人胚胎干细胞研究提供一个非常有用的细胞模型。方法 :通过Fugene 6基因转染试剂成功地将EGFP基因转入无饲养层培养的人胚胎干细胞中 ,嘌呤霉素筛选得到稳定表达EGFP的克隆 ;利用倒置荧光显微镜和流式细胞仪检测EGFP在人胚胎干细胞中的表达情况。结果 :EGFP瞬时转染效率为 30 %～ 40 % ,稳定转染效率约 1/10^4～5,且稳定转染的人胚胎干细胞均表达EGFP。结论 :Fugene 6是一种良好的基因转染试剂 ,它可以有效地将外源基因转入人胚胎干细胞中 ,为人胚胎干细胞的转基因研究提供新的实验手段。     

青鳉干细胞及其应用

干细胞广泛存在于多细胞生物的早期胚胎和成体组织中.干细胞的多能性和易操作性使其在发育生物学和再生医学上具有巨大的研究和应用价值,如细胞发育潜能的调控、细胞命运的决定、细胞治疗等.干细胞培养是研究干细胞研究工作的基础,主要集中在小鼠、人和青鳉这3种脊椎动物上.青鳉是一种小型淡水鱼,常被用作发育生物学和生物医学研究的模式物种.本文将主要介绍青鳉干细胞系及其应用.青鳉的MES1是除小鼠以外的第一个胚干细胞系;SG3是第一个成体精原干细胞系,可以在体外形成具有运动能力的精子;HX1是首个单倍体胚干细胞系,通过核移植技术,将该单倍体细胞的细胞核移植到未受精卵细胞中,得到第一个可育的半克隆动物霍利.这些突破使青鳉毫无疑问地成为干细胞研究的理想模式.     

表达红色荧光及转胸腺素β4基因绵羊胎儿成纤维细胞系的制备

旨在构建胸腺素β4(thymosin beta4,Tβ4)基因真核表达载体并转染绵羊胎儿成纤维细胞,获得稳定表达胸腺素β4及红色荧光蛋白的转基因细胞克隆。将克隆载体pMD19TT中的胸腺素β4基因亚克隆到表达载体pIRES2-DsRed2的多克隆位点,构建表达载体pIRES2-DsRed2-Tβ4,脂质体介导转染绵羊胎儿成纤维细胞,G418筛选获得稳定转染的细胞克隆。RT-PCR检测Tβ4基因在宿主细胞中的转录。测序结果显示,构建的表达载体pIRES2-DsRed2-Tβ4序列中,Tβ4基因正确连接在CMV启动子下游,顺序连接IRES2序列和红色荧光蛋白基因,载体构建正确。脂质体介导的稳定转染效率约为15%,经G418筛选得到转基因细胞克隆并高效表达红色荧光蛋白。RT-PCR检测显示外源Tβ4基因在绵羊胎儿成纤维细胞中得到转录。成功构建具有红色荧光蛋白和新霉素抗性双选择标记的胸腺素β4基因真核表达载体并稳定转染绵羊胎儿成纤维细胞,筛选得到的超表达胸腺素β4绵羊胎儿成纤维细胞系为下一步通过核移植和克隆技术获得转基因绵羊提供了条件。     

青鳉prdm14的原核表达、多克隆抗体制备及其应用

青鳉(Oryzias latipes)是研究遗传发育和细胞多能性的重要模式鱼类, 为探究prdm14同源基因的潜在作用, 实验将青鳉prmd14经原核表达后制备了兔抗Prdm14多克隆抗体。首先, 将prdm14基因的部分编码区连接到pET32a质粒中, 构建重组表达载体pET32a-prdm14?600。随后将重组载体转化至大肠杆菌(Escherichia coli)Rosetta(DE3), 经异丙基-β-d-硫代半乳糖苷(Isopropyl-β-d-thiogalactoside, IPTG)诱导表达, 获得分子量为60 kD的Prdm14重组蛋白。接着大量诱导蛋白表达并切胶纯化, 免疫家兔(Oryctolagus cuniculus), 6周后获得阳性抗体, 最后通过ELISA和Western blot检测抗体效价及其特异性。结果显示, 在37℃、0.6 mmol/L IPTG、诱导3h的条件下, 可获得Prdm14重组蛋白的高效表达; 制备的兔抗青鳉Prdm14多克隆抗体能够特异性识别青鳉组织中表达的Prdm14蛋白以及在HepG2细胞中过表达的青鳉Prdm14: EGFP融合蛋白。综上所述, 研究首次制备了一种能有效识别青鳉Prdm14的多克隆抗体, 该抗体的获得为后续研究prdm14基因在鱼类多能性干细胞中的作用提供了有力工具。     

基质结合区与转基因动物的基因表达

基质结合区(MAR)在稳定转染的细胞系中的研究结果显示,能缓冲在其侧翼的染色质某些拮抗作用.这为外源基因在染色体中随机整合的转基因动物研究提供了新的方向.文章对其在转基因动物中的探索性研究及可能的机理进行综述.指出在转基因动物中,MAR的应用能导致建立独立的基因活性结构域.它对基因高效表达无疑具有重要作用.MAR可能是一种新的顺式作用元件,与增强子、启动子协同作用调节基因的表达.     

α-HNP-1转基因细胞系的建立

目的构建稳定表达人α-HNP-1的转基因细胞系,为稳定生产α-HNP-1并将其应用于医药开发提供生产细胞源。方法真核表达载体pcDNA3.1(-)/HNP-1经酶切和测序鉴定后,用脂质体转染法转染昆明白小鼠胚胎干细胞来源的上皮细胞,通过不同浓度的G418加压筛选,建立稳定转染的胚胎干细胞来源的上皮细胞系,用RT-PCR及抑菌试验检测α-HNP-1的表达。结果建立了稳定转染的ES来源的上皮细胞系,成功地表达目的基因,其培养上清液及细胞冻融液具有抑菌作用,结论真核表达载体稳定转染胚胎干细胞来源的上皮细胞系,为进一步研究α-HNP-1的功能奠定了基础。     

Analysis of heterologous and homologous promoters and enhancers in vitro and in vivo by gene transfer into Japanese medaka (Oryzias latipes) Xiphophorus.

Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a number of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish cell lines and in embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoter-enhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. Fro the data obtained, a set of expression vectors for basic research as well as for aquaculture purposes were established.     

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.     

Medaka as a model of transgenic fish.

The medaka (Oryzias latipes) is an egg-laying fresh-water fish. We describe the medaka as a model system of transgenic fish in germs of biological characteristics, manipulation of embryos, gene expression in development, and basic research in aquaculture. The fish are small (approximately 3 cm in length) and have a short generation time (approximately 3 months). The eggs are easy to manipulate. A foreign gene (e.g., the chicken delta crystallin gene) is transferred and expressed stage-dependently in development of medaka embryos. Growth hormone genes of vertebrates are transferred and expressed and, in some cases, accelerate growth of the fish. Thus, the medaka is one of the most promising models of transgenic fish for basic research of gene expression and aquaculture.     

Liver receptor homologue-1 (LRH-1) activates the promoter of brain aromatase (cyp19a2) in a teleost fish, the medaka, Oryzias latipes

The medaka, Oryzias latipes, like other fish, have two distinct aromatase genes, the ovarian (cyp19a1) and brain (cyp19a2) forms. We previously reported that Ad4BP/SF-1, a member of the NR5A subfamily, plays an important role in the regulation of cyp19a1 expression in medaka ovarian follicles during vitellogenesis. In the present study, we investigated whether liver receptor homologue-1 (LRH-1), another NR5A subfamily member, is involved in the regulation of cyp19a2 expression in the medaka brain. In situ hybridization analysis revealed that LRH-1 was expressed in the hypothalamus, where it colocalized with aromatase (cyp19a2). We then showed by transient transfection assays that LRH-1 was able to increase expression of a cyp19a2 reporter gene in various mammalian cell lines, and that mutation of a putative LRH-1 binding site within the cyp19a2 promoter abolished this effect. Taken together, these findings suggest that LRH-1 plays a role in regulating cyp19a2 expression in the medaka brain. This is the first to demonstrate in vitro the activation of brain aromatase by LRH-1 in the vertebrate brain.     

Lack of ultraviolet-light inducibility of the medakafish (Oryzias latipes) tumor suppressor gene p53

P53 is by far the most frequently altered gene in mammalian tumors. However, so far not a single p53 lesion has been reported in malignancies of cancer model systems in lower vertebrates. For analyzing the function of p53 in lower vertebrates, the gene was cloned from the medakafish (Oryzias latipes). Despite some differences in the genomic organization, the fish p53 amino acid sequence is highly conserved. Contrary to higher vertebrates, the level of p53 mRNA in medaka embryos gradually increases during embryogenesis. High expression of the p53 mRNA was detected in melanoma cells compared to undetectable expression of the gene in embryonic stem cells and fibroblasts. No effect of ultraviolet (UV) irradiation on the expression of p53 in cell cultures as well as in medaka fry was observed, indicating a possible difference in the function of p53 in lower vertebrates.     

Mycobacterium marinum produces long-term chronic infections in medaka: a new animal model for studying human tuberculosis

Human infection by Mycobacterium tuberculosis is endemic, with approximately 2 billion infected and is the most common cause of adult death due to an infectious agent. Because of the slow growth rate of M. tuberculosis and risk to researchers, other species of Mycobacterium have been employed as alternative model systems to study human tuberculosis (TB). Mycobacterium marinum may be a good surrogate pathogen, conferring TB-like chronic infections in some fish. Medaka (Oryzias latipes) has been established for over five decades as a laboratory fish model for toxicology, genotoxicity, teratogenesis, carcinogenesis, classical genetics and embryology. We are investigating if medaka might also serve as a host for M. marinum in order to model human TB. We show that both acute and chronic infections are inducible in a dose dependent manner. Colonization of target organs and systemic granuloma formation has been demonstrated through the use of histology. M. marinum expressing green fluorescent protein (Gfp) was used to monitor bacterial colonization of these organs in fresh tissues as well as in intact animals. Moreover, we have employed the See-Through fish line, a variety of medaka devoid of major pigments, to monitor real-time disease progression, in living animals. We have also compared the susceptibility of another prominent fish model, zebrafish (Danio rerio), to our medaka-M. marinum model. We determined the course of infections in zebrafish is significantly more severe than in medaka. Together, these results indicate that the medaka-M. marinum model provides unique advantages for studying chronic mycobacteriosis.     

Chronic Mycobacterium marinum infection acts as a tumor promoter in Japanese Medaka (Oryzias latipes)

An accumulating body of research indicates there is an increased cancer risk associated with chronic infections. The genus Mycobacterium contains a number of species, including M. tuberculosis, which mount chronic infections and have been implicated in higher cancer risk. Several non-tuberculosis mycobacterial species, including M. marinum, are known to cause chronic infections in fish and like human tuberculosis, often go undetected. The elevated carcinogenic potential for fish colonies infected with Mycobacterium spp. could have far reaching implications because fish models are widely used to study human diseases. Japanese medaka (Oryzias latipes) is an established laboratory fish model for toxicology, mutagenesis, and carcinogenesis; and produces a chronic tuberculosis-like disease when infected by M. marinum. We examined the role that chronic mycobacterial infections play in cancer risk for medaka. Experimental M. marinum infections of medaka alone did not increase the mutational loads or proliferative lesion incidence in all tissues examined. However, we showed that chronic M. marinum infections increased hepatocellular proliferative lesions in fish also exposed to low doses of the mutagen benzo[a]pyrene. These results indicate that chronic mycobacterial infections of medaka are acting as tumor promoters and thereby suggest increased human risks for cancer promotion in human populations burdened with chronic tuberculosis infections.