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.     

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。     

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。     

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.     

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。     

一种通过体表注射斑马鱼性腺的简易投药方法

验证了一种简便易行的斑马鱼性腺体表注射方法。首先解剖10条成年斑马鱼,精确测量并勾画出性腺的综合体表投影;然后注射阿尔新蓝染料确定注射路径和范围;再分别注射葡聚糖德克萨斯红和Sox9amyc过表达质粒与脂质体转染试剂混合物,48 h后分别用荧光显微镜和免疫印迹法检测荧光分布和融合基因表达。根据雄性斑马鱼平均体长、体宽和性腺的平均长宽,确定注射点位于斑马鱼胸鳍上端水平线与腹鳍前端竖直线相交处向上0.1 cm处。注射48 h后,发现阿尔新蓝和葡聚糖德克萨斯红多存在于性腺内部,部分标记了周围器官边缘部位;证实Sox9a-myc蛋白在卵巢中大量表达。结果表明建立的斑马鱼性腺体表投影体侧注射方法有效可行,为以后斑马鱼的体内基因调控研究提供了简便易行的给药途径。     

利用CRISPR/Cas9系统高效敲除斑马鱼lncRNA基因启动子区

斑马鱼(Danio rerio)长非编码RNA(long non-coding RNA,lncRNA)基因Nondret002679与人(Homo sapiens)肿瘤相关基因间长非编码RNA682基因LNC-PHOX2B-2具有同源序列.本研究以斑马鱼为模型,利用成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列关联蛋白9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9,CRISPR/Cas9)系统,敲除预测的Nondret002679基因启动子区以沉默Nondret0026 79基因的表达,研究IncRNA基因Nondret0026 79在斑马鱼中的调控功能.首先利用启动子区上下游序列设计靶向不同位点的向导RNA(guide RNA) gR1、gR2、gR3、gR4、gR5和gR6.人工合成gRNA转录模板,体外转录为gRNA,与Cas9 mRNA一起显微注射到斑马鱼卵中.PCR鉴定28尾2月龄斑马鱼发现,11尾发生敲除,敲除率为39％.繁殖启动子区敲除的杂合鱼,获得33尾F1代,经鉴定有6尾F1斑马鱼启动子区缺失,其中3尾缺失是在两条同源染色体上.该初步研究表明,CRISPR/Cas9系统可高效敲除lncRNA基因的启动子区,并且这种敲除可以遗传至下一代,为研究lncRNA功能提供了一个有效的基因编辑工具.     

洛克沙胂暴露对斑马鱼的急性毒性及其组织中HSP70的表达定位

[目的]对洛克沙胂的水生生态安全评价、畜禽粪便管理以及HSP70在斑马鱼应激状态下的保护作用的研究提供科学依据。[方法]以斑马鱼为试验生物,进行洛克沙胂暴露对斑马鱼96 h的急性毒性试验,并对洛克沙胂暴露下斑马鱼组织（鳃、肝脏、性腺）中HSP70的表达定位进行了免疫组织化学初步研究。[结果]洛克沙胂对斑马鱼的急性毒性较低,96 hLC50为249.80 mg/L;在浓度100mg/L洛克沙胂暴露下,HSP70在斑马鱼的鳃、肝脏、性腺3种组织中有特异性表达,主要定位在鳃小片的基底膜、鳃弓和鳃丝上皮细胞及红细胞的胞质中,性腺的基质细胞中,肝脏的静脉血管四周和红细胞胞质中。[结论]洛克沙胂对于斑马鱼的毒性属于低毒范围。     

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.