Open-design Recirculating Systems for Zebrafish Culture

The zebrafish is an established vertebrate-animal model in biomedical research. Currently, their mass culture is mainly done using systems provided by commercial suppliers. Commercial systems are compact, recirculating, and use auto-cleaning tanks. These features minimize space use, labour cost, and water wastage; thus, facilitating maintenance of a large number of zebrafish using minimal resources. However, the often considered costs associated with these systems often impose a barrier to current and prospective researchers, especially those with limited funds or working in labs with no access to institutional centralized zebrafish culture facilities. In contrast to commercial systems, custom-made zebrafish maintenance systems are also described in the literature. To distinguish custom-made system from commercial systems, we termed them as “open-design” systems. Open-design systems are cost-effective, modular, and frequently being improved by zebrafish researchers. However, for further development and to present them as a viable option for zebrafish researchers around the world, a review of their current status and technical understanding is required. Here, we compile the disparate data on the purpose and technological development of the open-design systems. We believe this review will be a valuable resource for all zebrafish users and help streamline the open-design technology for zebrafish culture.     

毒死蜱对斑马鱼血清CAT和SOD活性的影响

通过对斑马鱼肌肉注射不同浓度(2、20、200、2000μg/kg)的毒死蜱,研究了斑马鱼血清中过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性的变化情况。结果显示:在处理后24 h两种酶的活性均明显升高;在处理后96 h两种酶的活性均显著下降。说明毒死蜱对斑马鱼机体的抗氧化系统有明显的损伤作用。     

用斑马鱼检测猪链球菌2型的致病力

 【目的】猪链球菌2型（Streptococcus suis type 2，SS2）菌株致病力各异，以斑马鱼为实验动物，建立了较为简便可靠的SS2致病力检测方法。【方法】选用1 005尾AB系斑马鱼（Danio rerio）以检测SS2不同分离株的致病力。检测8株基因型均为mrp+ef+、对猪有致病力的SS2菌株。【结果】结果斑马鱼接种菌株12 h后呈败血症病变，96 h内对斑马鱼的半数致死量（LD50）在5.36×103至5.01×104 cfu之间。上述接种菌株的斑马鱼均从体内重新分离到接种菌。同时检测1株基因型为mrp-ef-、对猪无致病力的SS2菌株，斑马鱼接种后96 h内不表现任何病变，亦不出现死亡，对斑马鱼的LD50>106 cfu。SS2有毒力株和无毒力株对斑马鱼的LD50差异极显著（P＜0.01）。【结论】斑马鱼可作为研究猪链球菌2型菌株感染的动物模型。     

Effect of lead exposure on mRNA expression of NMDA receptors in zebrafish embryos and larvae

AIM:To investigate the effect of lead exposure on mRNA expression of N-methyl-D-aspartate(NMDA) receptors in zebrafish embryos and larvae. METHODS:Zebrafish embryos(wild type; AB line) were exposed to lead acetate(PbAc) at concentrations of 0, 0.1, 0.5, 2.5 and 12.5 μmol/L, respectively. Total RNA was extracted from zebrafish embryos or larvae at the time points of 24, 48, 72, 96 and 120 hours post fertilization(hpf). The mRNA levels of NR1.1, NR1.2 and NR2B were determined by real-time quantitative PCR. RESULTS:The mRNA expression of NR1.1, NR1.2 and NR2B gradually increased during embryonic development, raised rapidly at 72 hpf, peaked at 96 hpf(vs that at 24 hpf, P<0.01), and still kept in high level at 120 hpf in control group. The impact of lead exposure on the mRNA expression of NR1.1, NR1.2 and NR2B varied with lead concentrations. With the increasing concentrations of PbAc, the mRNA expression of NR1.1 generally increased and reached the highest level ahead of 96 hpf. The peak mRNA level of NR1.1 was observed at 72 hpf under the condition of PbAc exposure at the concentrations of 2.5 and 12.5 μmol/L, and were higher than that in control group(P<0.05). Similarly, the mRNA levels of NR1.2 and NR2B showed an increasing trend with PbAc exposure. However, the peaking time of NR1.2 and NR2B in mRNA expression spanned from 72 to 120 hpf. The significant correlations between the expression levels of NR1.1, NR1.2 and NR2B were observed(P<0.01) and the Pearson’s correlation coefficient values of r_NR1.1-1.2, r_NR1.1-2B and r_NR1.2-2B were 0.681, 0.637 and 0.514, respectively. CONCLUSION:The mRNA expression of NR1.1, NR1.2 and NR2B gradually increases throughout the embryonic development of zebrafish and reaches the highest levels at early stage of larva. Close correlations between the mRNA expression of NR1.1, NR1.2 and NR2B are present during the period of embryo and larva. Lead exposure induces up-regulation and forward shift of NR1.1, NR1.2 and NR2B at mRNA level, indicating that lead induces abnormal expression of NMDA receptors.     

斑马鱼不同发育时期血管内皮生长因子受体2基因表达的实时定量PCR分析

[目的]探讨血管内皮生长因子受体2（VEGFR2-）基因在斑马鱼不同发育时期的表达。[方法]分别从12、24、48、72和96 h的斑马鱼胚胎和仔鱼中提取总RNA,用实时定量RT-PCR方法检测VEGFR2-基因表达,采用2^-△△Ct法进行数据分析。[结果]VEGFR2-基因表达量在12-72 h呈上升趋势,在96 h有所下降。12 h表达量最低,72 h表达量最高,与其他发育期均有显著差异。[结论]斑马鱼血管发育至72 h达成熟阶段。血管发育成熟前,VEGFR2-基因表达水平逐步增长;血管发育成熟后,VEGFR2-基因表达水平下降。     

TLR21在斑马鱼免疫功能中的作用研究

［目的］研究斑马鱼TLR21受体家族的作用。［方法］用各种纯组分刺激斑马鱼,研究斑马鱼TLR21受体家族（zTLR21.1、zTLR21.3、zTLR21.4）的表达量变化,推测它们的功能。［结果］zTLR21.1对于LTA刺激无反应,对于LPS、ployI：C和Glucan刺激都有上调反应。zTLR21.3和zTLR21.4对于LPS、LTA和ployI：C刺激均无反应,对于Glucan刺激有明显上调变化。［结论］zTLR21.1可能是一种能够识别大部分抗原的模式识别受体。zTLR21.3和zTLR21.4的功能很类似,可能都是酵母的特异识别受体。     

转座子介导细胞质显微注射技术的基因转移效率比较

为了比较和探索SB(sleeping beauty)、PB(piggyBac)和T012 3种转座子介导下的细胞质显微注射法在小鼠(Mus musculus)和斑马鱼(Danio rerio)上的转基因效率,本研究将包含3种转座子的重组载体pT3-PST-CAG-GFP分别与3种转座酶表达载体以1:1质量比混合,显微注射至小鼠和斑马鱼受精卵细胞质,经体外培养后在胚胎发育不同时期用荧光显微镜检测报告基因绿色荧光蛋白(green fluorescent protein,GFP)阳性率,结果显示,在培养至4 Ed(96 h)(Ed: embryo day,胚胎发育的天数)的小鼠胚胎中,PB转座子介导的GFP转基因阳性率最高,达52％(N=281),显著高于SB的47％ (N=328)和To12的36％ (N=273)(P＜0.05);而受精后36和60 h (hour past fertilization,hpf)斑马鱼胚胎中,荧光显微镜检测表明,To12的GFP阳性率分别为36％(N=677)和37％(N=685),显著高于SB(28％ N=691和27％ N=708)和PB(30％ N=687和32％ N=675)(P＜0.05);转座子介导的细胞质显微注射可获得较高的转基因效率,且转座子活性具有物种差异性,在哺乳动物胚胎中PB转座子的基因转移效率最高,而在鱼类胚胎中To12转座子的基因转移效率最高.本研究为提高动物转基因效率研究提供重要参考资料.     

炔雌醇和菲2种环境内分泌干扰物对斑马鱼的影响研究进展

炔雌醇和菲在各种水环境中广泛分布,其对水生动物的繁殖、发育和生长等各方面有显著的影响.斑马鱼容易饲育,遗传学工具成熟,是研究脊椎动物的一种模式动物.综述了炔雌醇和菲2种内分泌干扰物对斑马鱼发育的影响及其机制的研究进展.     

CRH、UⅠ和UⅡ基因在斑马鱼发育早期的表达谱

促肾上腺皮质激素释放激素(Corticotropin-releasing hormone,CRH)、尾加压素Ⅰ(UrotensinⅠ,UⅠ)和尾加压素Ⅱ(UrotensinⅡ,UⅡ)广泛存在于脊椎动物中。在鱼类中,它们在渗透压调节、繁殖、营养代谢以及应激反应中发挥着重要作用。本研究中,通过收集0、1.5、3.7、5.3、8、10、16、24、48、72、96、120和144 hpf(hours post-fertilization)的斑马鱼早期发育样品,运用荧光定量PCR技术进行检测发现CRH、UⅠ、UⅡα和UⅡβmRNA从0 hpf开始存在表达,而且CRH、UⅠ、UⅡα和UⅡβ的表达量随发育而逐渐增加,并分别在120 hpf或144 hpf达到最高水平。相关性分析揭示在斑马鱼早期发育过程中,CRH、UⅠ、UⅡα和UⅡβmRNA的表达水平两两之间呈现极显著正相关,这表明CRH、UⅠ、UⅡα和UⅡβ在斑马鱼早期发育过程中关系紧密并且相互合作发挥作用。     

斑马鱼胰腺发育的研究进展

准确理解发育过程中的分子事件从而调控胰腺发育,对临床医学相关的糖尿病、肥胖、胰腺癌和疾病有着重要影响。1996年以前,胰腺发育和分化研究的主要动物模型是老鼠和与早期发育相关的鸡和爪蟾。斑马鱼作为一个新的研究模型,通过参与功能基因组学、遗传学和体内全胚胎观察,斑马鱼已经大范围应用和基因功能的多维度定义在胰腺的形成和分化过程中。主要介绍了模式动物斑马鱼早期胰腺发育和形态建成。