大菱鲆受精卵显微注射技术的建立

本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein,GFP)基因的重组质粒(smyd1:gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1:gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。     

显微注射技术在制备鱼类嵌合体和转基因海水鱼上的应用

以鱼类胚胎细胞和胚胎干细胞为核供体进行细胞移植构建鱼类嵌合体研究方面,现有的成功报道均采用显微注射方法;在转基因海水鱼类研究中,显微注射也是最为常用的技术,本实验室在花鲈胚胎干细胞嵌合体构建和外源基因向花鲈胚胎的转移研究中取得的结果也充分证实,显微注射技术是开展海水鱼类细胞移植和转基因研究的首选技术。     

Liposome‐mediated messenger RNA: An alternative for fish cell transfection in culture

Liposome‐mediated plasmid DNA (pDNA) transfection is a relatively efficient transfection method that depends highly on actively dividing cells. However, numerous fish cells are mitotically inactive under in vitro culture conditions, thus limiting the application of liposome‐mediated pDNA transfection. Liposome‐mediated mRNA transfection is an attractive alternative in post‐mitotic cells because it eliminates the transfer of genes into the nucleus and therefore is independent of cell proliferation. In this study, pDNA is compared with mRNA encoding biomarker proteins (enhanced green fluorescent protein [eGFP] and luciferase) mediated by cationic liposome in zebrafish embryo fibroblast cell line (ZF4), with or without proliferation restriction, and in mitotically inactive turbot liver primary cells. The results indicated that mRNA encoding eGFP exerted much higher transfection efficiency than pDNA in ZF4 cells. Moreover, mRNA encoding eGFP was detected and disappeared sooner than pDNA. Chemiluminescence of the biomarker protein luciferase showed that liposome‐mediated mRNA transfection translated about threefold more protein without proliferation restriction and about tenfold more protein with proliferation restriction than pDNA transfection. In turbot liver cells, mRNA induced higher transfection efficiency and greater luciferase protein expression than pDNA. Greater PPARα expression and expression of downstream target genes were induced by mRNA rather than by pDNA transfection. In conclusion, cationic liposome‐mediated mRNA is an alternative for fish cell transfection due to higher transfection efficiency and protein expression levels and faster translation onset.     

Construction of an expression vector containing a β-actin promoter region for gene transfer by microinjection in red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   Transgenic technology has been widely applied to a variety of freshwater fish species. However there are few reports on the use of this technology in commercially important marine species. In this study, the construction of expression vectors containing the β-actin promoter region for use in the red sea bream Pagrus major , a species of considerable importance to the aquaculture industry in Japan is reported. The β-actin gene was cloned from a red sea bream genomic DNA library. Recombinant plasmids were constructed by linking the 5' flanking region of the β-actin gene to the green fluorescent protein reporter gene, followed by the poly A signal sequence of simian virus 40 or the 3' flanking region the β-actin gene. Expression of these constructs was examined following microinjection into zebrafish and red sea bream embryos, and compared to that of the expression vector pXI-GFP driven by the Xenopus elongation factor 1α. The results indicated that the construct consisting of the β-actin 5'-and 3' flanking regions was the most efficacious. In future studies, it is planned to investigate the efficient condition for integration into chromosomes of the transgene.     

Studies on chorion hardening inhibition and dechorionization in turbot embryos

Embryo dechorionization is a common practice used in certain fish species for different purposes. It facilitates techniques like microinjection, transfection or electroporation in embryos. Dechorionization is easily achieved in some fish species but is a more complex problem in species that have very thick chorions. In this study, we address this problem in turbot embryos, where chorion removal is practically unachievable post-chorion hardening. For this purpose, different solutions that lacked ions required for the hardening of this envelope or contained inhibitors of enzymes involved in the process were used during egg fertilization. The toxicity of the solutions was assessed, and their effect on embryo cleavage and on chorion structure was studied by light and scanning electron microscopy (SEM). The results demonstrated that embryos are very sensitive to these solutions and that first cellular cleavages are affected with most of them. This study also provides the first report on turbot chorion structure, analyzed by SEM. The chorion is a very thick envelope in this species, and its total removal was not observed with the employed treatments. Nevertheless, partial dechorionization was achieved when embryos were fertilized in some of the tested solutions and later treated with pronase (3 mg/ml).     

Pectin purified from pomelo (Citrus maxima) peel as a natural hatching agent for fish embryos

Pectin is a biodegradable polysaccharide, and it has been recently applied as a gene delivery, drug delivery, wound healing and tissue engineering agent. In this study, pectin was extracted from pomelo (Citrus maxima) peel and characterized. The extraction recovery of pectin form pomelo peel was 14.5%, and it had 72.56% degree of esterification, 1,245.56 equivalent weight, 7.82% methoxyl and 68.27% anhydrouronic acid contents. Use of pomelo pectin as a hatching enhancing agent for fish embryos and its effect on hatching enzyme 1 (ZHE1) was investigated. The pectin‐exposed zebrafish embryos (100 µg/ml) showed significantly (p < .05) higher hatching rate (96.6%) compared with untreated (control) embryos (66.6%) at 60 hpf. The mRNA expression of ZHE1 was also significantly (p < .05) elevated up to 55.6‐fold in pectin‐exposed embryos at 24 hpf. In situ hybridization results revealed remarkably strong expression of ZHE1 in pectin‐exposed embryos compared with the control group. In addition, considerably larger size of the hatching gland was observed in pectin‐exposed larvae than that of the unexposed larvae group. These results clearly indicate that pectin isolated from pomelo peel has an ability to enhance the hatching process of zebrafish embryos via upregulation of ZHE1.     

Microinjection of the antifreeze protein type III (AFPIII) in turbot (Scophthalmus maximus) embryos: Toxicity and protein distribution

Fish embryo cryopreservation has not been achieved. Different methods and alternative cryoprotective agents (CPAs) should be explored in order to succeed in this purpose. Antifreeze proteins (AFPs) are naturally expressed in sub-arctic fish species, and they inhibit the growth of ice crystals as well as recrystallization during thawing. Therefore, their introduction into embryos can be highly beneficial for vitrification purposes. In this study, AFP type III was introduced into turbot embryos, by microinjection into the yolk sac and the perivitelline space at F stage (tail bud).Toxicity and distribution of protein in microinjected embryos were established before testing the protein effect on embryo cryopreservation. AFP-FITC distribution within the embryo was analyzed by confocal microscopy at 5 min and 24 h after microinjection in F stage embryos. To test the sensitivity of microinjected embryos to CPAs, embryos were subjected to a protocol for the incorporation of a vitrifying solution that was specially designed for turbot embryos. Hatching rates after CPA incorporation were determined. Results indicate that embryos at late developmental stages are more resilient to microinjection, with embryo survival rates between 60 and 82%. Confocal microscopic images demonstrated that the protein was homogeneously distributed within the microinjected embryo compartment, but did not enter any other compartment. On the other hand, microinjected embryos successfully surmounted their incubation in the CPAs. This study explores new alternatives for cryopreservation suggesting the use of natural cryoprotectants (AFPs) in the protection of intra-embryo compartments, which are usually unprotected with the conventional cryopreservation protocols for fish embryos.     

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron‐like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1‐TRBIV‐MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT‐PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT‐PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post‐vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon‐induced antiviral molecules were observed in the vaccinated fish 60 h post‐vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.     

Effect of dietary common carp by‐product protein hydrolysates on antioxidant status in different organs of zebrafish (Danio rerio)

A feeding trial was conducted to evaluate the effect of dietary protein hydrolysates from common carp (Cyprinus carpio) by‐products on the antioxidant status of zebrafish (Danio rerio). Common carp by‐product was hydrolysed using Alcalase to a degree of hydrolysis of 15%. The zebrafish were fed for 44 days with four different diets with increasing levels of carp by‐product hydrolysates (CBH0: 0 g/kg; CBH25: 25 g/kg; CBH50: 50 g/kg; CBH100: 100 g/kg). The gills, muscle and brain were dissected at the end of the feeding trial in order to evaluate the total antioxidant capacity against peroxyl radicals (ACAP) and lipid peroxidation (TBARS). Although total antioxidant capacity did not show differences in muscle (p > 0.05), lipid peroxidation was reduced in the muscle of fish fed the CBH50 diet. Brain lipid peroxidation showed a significant reduction (p < 0.05) in all groups when compared with the control diet CBH0. Antioxidant properties of protein hydrolysates indicate their potential as nutraceuticals since (a) a reduction in muscle lipid peroxidation was verified, implying that their use could enhance the quality and shelf life of fish fillets; and (b) a decrease in brain lipid peroxidation was registered, highlighting the potential use of fish protein hydrolysates for the prevention of neurodegenerative diseases.     

Dietary oils influence ovary and carcass composition and embryonic development of zebrafish

To evaluate the effect of different dietary lipids on zebrafish reproduction, we examined parameters including ovary and carcass composition, oestradiol (E2) levels, eclosion rate and embryonic development. In our study, zebrafish were subjected to a 5‐month feeding trial whereby olive (OLV), linseed (LIN), fish (FIS) or corn (CRN) oil were used to compose four isonitrogenous and isoenergetic diets. A positive correlation was found between ovary EPA and whole‐body E2 concentration (P = 0.005). The developmental dynamics was affected by dietary lipids and embryos from CRN treatment females; which had higher percentage of ARA in ovary (1.48 ± 0.44%) (P = 0.015), developed faster at 8–9 h postfertilization (hpf) than the ones originated from females receiving other diets (P = 0.0069). This effect was not sustained during later observation periods, suggesting ARA may act as a modulator of developmental dynamics only during initial phases. There was no clear effect of dietary lipid source on ovary protein content (P = 0.304) and eclosion rates at 72 hpf (P = 0.0623). Dietary fatty acids play an important role in reproductive outcome; however, additional studies are necessary to elucidate the mechanisms by which HUFA affect embryonic development.     

Evaluation of growth performance and lipid metabolism in zebrafish fed fructooligosaccharide using RNA sequencing

This study evaluated the effects of fructooligosaccharide (FOS) on growth performance and lipid metabolism in zebrafish (Danio rerio) by RNA sequencing. A total of 240 healthy zebrafish were randomly distributed into two groups. The control group was fed a basal diet, and the treatment group was fed a basal diet supplemented with 0.4% FOS. The results showed that there was no significant difference in growth performance (p > 0.05). The lipid content, total cholesterol, triglyceride, free fatty acid and low‐density lipoprotein were significantly lower (p < 0.05) in the liver of fish fed the 0.4% FOS diet than those of the fish fed the control diet, while the fish fed the 0.4% FOS diet had significantly higher (p < 0.05) high‐density lipoproteins than those of the control fish. Malic enzyme and fatty acid synthetase activities were significantly reduced by adding FOS. KEGG pathway analysis showed that the two pathways of steroid hormone biosynthesis and steroid biosynthesis were significantly enriched (p < 0.05). The profile of genes in these two pathways was affected by FOS in zebrafish. The results of the two pathways suggested new mechanisms underlying the lipid metabolism mechanism of FOS.     

In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation

Micro (mi)RNAs are abundant small noncoding RNAs found in plants and animals, the regulatory functions of which are not fully understood in fish. To identify potential miRNAs, we screened an miRNA microarray with total RNA from zebrafish infected with Vibrio harveyi and another from uninfected zebrafish. Six miRNAs were obtained from the microarray screening. We studied miRNA expression patterns of 2 miRNAs (miR-122 and miR-194) after bacterial infection of transgenic zebrafish (containing tilapia hepcidin (TH)2-3) and non-transgenic zebrafish from which the 2 miRNAs were obtained from the microarray experiment. The results indicated that miR-122 and miR-194 were higher in PBS-injected zebrafish compared with TH2-3 zebrafish or wild-type (WT) zebrafish after V. harveyi infection. Overexpression of miRNAs (miR-122, miR-192, and miR-194a) was seen in zebrafish liver (ZFL) cells after lipopolysaccharide (LPS) treatment and in untreated fish. Our results showed that after 24?h of doxycycline treatment without LPS stimulation, interleukin (IL)-22, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, and tumor necrosis factor (TNF)-α gene expressions were, respectively, upregulated by ~14-, 22-, 2.2-, 13-, 200-, and 38-fold in miR-122-transfected compared with non-transfected (WT) ZFL cells. In cells transfected with miR-192 and treated with LPS after 8-12?h, IL-22, lysozyme, TLR1, TLR3, TLR4a, and TNF-α expressions significantly differed between WT and miR-192-overexpressing ZFL cells. However, we observed significantly higher IL-22 expression levels after 12?h of LPS treatment in miR-192-transfected ZFL cells compared with non-transfected cells. In contrast, IL-22, lysozyme, and TNF-α were markedly upregulated (>100-fold) after miR-194a transfection and overexpression in ZFL cells and treatment with LPS. Our cloning and expression analyses indicated that miR-122, miR-192, and miR-194a play important roles in zebrafish immunology.     

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host–pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV‐1) and cyprinid herpesvirus 3 (CyHV‐3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up‐regulated the expression of antiviral genes vig‐1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV‐3 induce up‐regulation of vig‐1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV‐3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV‐infected fish, a prolonged confrontation of the host with the pathogen could be studied.     

Procedural Protocol, Survival to Hatching and Plasmid DNA Fate After Microinjection into Tilapia Zygotes

The plasmid vector, pBRd-AK1-BGH4.6.10 (pBGH), containing the bovine growth hormone sequence driven by an avian retroviral long terminal repeat (LTR) was microinjected at 0.1, 0.5, 1 or 5 ng of pDNA/20 nl of physiological saline into tilapia, Oreochromis mossambicus × O. niloticus embryos. In 24 replicates, normally 60 zygotes were microinjected with either the entire linear 8.5 kb pBGH/ClaI vector or a 3.8 kb pBGH/SalI restriction fragment. Overall mean survival to fry hatching was 7.6% in plasmid DNA-microinjected, 15.6% in sham-microinjected and 48.1% in uninjected control embryos. There were no significant ( P > 0.05) differences in survival to hatching between those embryos microinjected with the 3.8 or the 8.5 kb restriction fragment, nor was there a trend toward decreasing survival as the plasmid DNA concentration increased from 0.1 to 5 ng. The significant ( P < 0.05) increase in survival among uninjected control embryos to hatching indicates that microinjection trauma was the major cause of mortality. Large quantities of plasmid DNA were recovered from pooled-embryo samples. Multiple bands (positive signals) were detected usually in the high molecular weight (HMW) genomic DNA regions. Position shifting of these HMW bands upon digesting with various restriction endonucleases provided evidence for plasmid DNA integration into tilapia embryo chromosomal DNA. Otherwise, these positive signah may have been end-twnd ligations of increasingly longer plasmid DNA constructs. Putative transgenic O. mossmbicus × O. niloticus were found among eight of 27 surviving adults.     

Detection and partial characterization of a UV-damaged-DNA binding activity highly expressed in zebrafish (Danio rerio) embryos

The expression of distorted DNA-binding factors was studied in developing zebrafish (Danio rerio) using UV-damaged DNA as the binding target. A strong and high-shifting binding activity was detected in the extracts of zebrafish early embryos (12 h after fertilization), and the expression of this activity dramatically decreased in 60 to 84-h-old zebrafish. The embryonic extracts produced a similar pattern of high-shifting complexes after incubating with a CPD-specific or a 6-4PP-specific probe, while different types of low-shifting complexes were generated by the extracts of 84-h-old larvae. The formation of high-shifting complexes was suppressed in the presence of NaCl at 0.25 M or higher concentrations, yet the production of low-shifting complexes was stimulated by increasing salt concentration. The binding activity expressed in zebrafish embryos was apparently unrelated to NER-associated damage-recognition protein XPA, since two polypeptides recognized by an anti-human XPA antibody were detected only in 84-h-old zebrafish extracts. A competitive binding assay indicated that both CPDs and 6-4PPs were recognized by the same binding activity expressed in 12-h-old zebrafish, and this activity contained at least two protein fractions that were eluted from a DEAE-cellulose column by NaCl at 0.1 M and 0.2 M. UV crosslinking of the two NaCl eluates to a 6-4PP probe produced covalent complexes with the same electrophoretic mobility except one 34-kDa complex generated by the 0.1 M NaCl eluate, suggesting the existence of two multisubunit damage-recognition protein complexes in zebrafish embryos. UV-binding factors found in 12-h-old zebrafish embryos may be involved in processing developmental stage-specific DNA structures similar to UV-damaged DNA. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dietary taurine supplementation in plant protein based diets do not affect growth and reproductive performance of zebrafish

Taurine (Tau) has been regarded as a conditional essential nutrient for some fish species. Although its role has been extensively studied in higher vertebrates, limited results are reported with fish especially its role on reproductive performance and the ontogenic changes on Tau levels throughout the life cycle. Therefore, we designed a feeding trial using zebrafish as a model species to test whether Tau supplementation to plant protein diets would have a positive effect on growth and reproductive performance. Zebrafish were fed plant protein diets containing graded levels of Tau (0.2, 4.6, 5.9 and 13.7 g/kg diet) from 10 days post fertilization (dpf) to sexual maturity. An additional commercial diet was used as a positive control for performance. The trial followed a completely randomized design with five treatments (diets) and three replications. After 60 days of feeding, growth, Tau concentration in the body, redox status, lipid body composition, reproductive and offspring performances were analysed. Tau supplementation did not affect growth and/or reproductive performance; however, zebrafish seems to differently modulate Tau concentration according to the growth stage. Tau seemed to induce a hypolipidemic effect in zebrafish by reducing lipid accumulation in their bodies (p < .05). A trend to a more pro‐oxidant effect of Tau supplementation was observed by the decreased reduced glutathione levels. In sum, Tau does not affect growth and reproductive performance of zebrafish but it is important for normal lipid utilization and redox status.     

Embryonic stem cells in fish: current status and perspectives

Totipotent embryonic stem (ES) cells represent a bridge that links in vitro and in vivo manipulations of animal genomes and have enormous potential for genetic engineering of livestock. We have recently established feeder cell-free conditions for culturing cells of midblastula embryos (MBE) of the medaka (Oryzias latipes) and obtained several stable cell lines that show all features of mouse ES cells in vitro. One of these lines, MES1, has been demonstrated to retain a diploid karyotype and can be induced to differentiate into various cell types in vitro. Upon microinjection into albino host blastulae, MES1 cells are able to form pigmented chimeras. Genotype-specific PCR analysis revealed that 90% of host blastulae transplanted with MES1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. Transplantation of genetically labelled MES1 cells revealed a wide contribution to numerous organs derived from all three germ layers and differentiation into various types of functional cells. These ES properties of MES1 line was not abolished by stable gene transfer and long-term selection. Thus MES1 cells may represent a first promising cellular vehicle for the production of genetically modified fish. The genetic background has been found to have a profound effect on the efficacy of ES cell derivation and of chimera formation.     

HSP90抑制剂根赤壳菌素对斑马鱼胚胎发育的影响

为研究热休克蛋白90(heat shock protein 90,HSP90)在斑马鱼(Danio rerio)胚胎发育中的作用,本实验采用2μmol/L、5μmol/L、10μmol/L的HSP90抑制剂根赤壳菌素(radicicol)对斑马鱼发育期胚胎进行处理,监测斑马鱼胚胎不同发育时期两个HSP90功能抑制标志基因BAG3(BCL2-associated athanogene3)和HSPB1(heat shock protein beta-1)的mRNA的表达水平,并观察不同发育时期的胚胎发育状况。结果如下:(1)实时荧光定量PCR结果显示,BAG3和HSPB1 mRNA水平在根赤壳菌素处理胚胎12 hpf(hours post-fertilization,hpf)或24 hpf后显著增高,Western blot检测到5μmol/L根赤壳菌素处理胚胎24 hpf后HSP70表达上调,证明该实验条件下HSP90功能受到抑制;(2)根赤壳菌素处理后斑马鱼胚胎发育变缓,胚胎成活率统计显示:2μmol/L、5μmol/L、10μmol/L根赤壳菌素处理24 hpf胚胎成活率分别是95%、77%、35%,成活率随根赤壳菌素浓度的增高而降低;(3)5μmol/L根赤壳菌素处理72 hpf可见部分个体色素沉积减少、心包膜增大、肌肉萎缩等发育畸形。研究结果证明HSP90在斑马鱼胚胎发育过程中发挥重要的作用,根赤壳菌素在5μmol/L时已达到较佳抑制效果且成活率较高,而且胚胎发育出现了各种形态学变化,为后期研究HSP90调节斑马鱼胚胎发育奠定了基础。