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Now showing items 17 - 32 of 94

  • A controllable on-off strategy for the reproductive containment of fish.

    Zhang, Yunsheng   Chen, Ji   Cui, Xiaojuan   Luo, Daji   Xia, Hui   Dai, Jun   Zhu, Zuoyan   Hu, Wei  

    A major impediment to the commercialization and cultivation of transgenic fish is the potential ecological risks they pose to natural environments: a problem that could be solved by the production of sterile transgenic fish. Here, we have developed an on-off reproductive containment strategy for fish that renders the offspring sterile but leaves their parents fertile. TG1 (Tol2-CMV-GFP-pA-CMV-gal4-pA-Tol2) and TG2 (Tol2-CMV-RFP-pA-5 * UAS-as/dnd-pA-Tol2) zebrafish lines were established using a GAL4/UAS system. While the parental lines remained fertile, in the hybrid offspring, GAL4 induced 5 * UAS to drive the transcription of antisense dnd, which significantly down-regulated endogenous dnd expression. This disrupted the migration of primordial germ cells (PGCs), led to their apoptosis, and resulted in few or no PGCs migrating to the genital ridge. This process induced sterility or reduced fertility in adult fish. This on-off strategy is a potentially effective means of generating sterile fish for commercialization while retaining fertility in brood stocks, and offers a novel method to mitigate the ecological risks of fish introductions. =20
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  • Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

    Su, Jianguo   Yang, Chunrong   Zhu, Zuoyan   Wang, Yaping   Jang, Songhun   Liao, Lanjie  

    In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P < 0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases. (C) 2008 Published by Elsevier Ltd.
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  • Growth hormone transgene effects on growth performance are inconsistent among offspring derived from different homozygous transgenic common carp (Cyprinus carpio L.)

    Zhong, Chengrong   Song, Yanlong   Wang, Yaping   Li, Yongming   Liao, Lanjie   Xie, Shouqi   Zhu, Zuoyan   Hu, Wei  

    Higher productivity of cultivated fish is required to sustain the supply of fisheries products in the light of increasing demand. We established different lines of homozygous growth hormone (GH) transgenic common carp and analyzed their biological characteristics. Comparing transgenic and control larvae derived from the same control fish mother, we found there was no significant difference in fertilization and hatch rates, but that hatch timing was advanced and average total length was significantly longer in transgenic carp. Early growth performance and other biological characteristics were compared among homozygous, hemizygous and control fish. Both the #TG2 and #TG3 lines exhibited greater average body weight, specific growth rate (SGR), feed conversion efficiency (FCE) and protein retention efficiency (PRE), as well as lower feeding rate (FR) and lipid and energy content than control fish. The energy retention efficiency (ERE) was significantly lower in the #TG3 line, while no difference was observed between the #TG2 line and control fish. These results indicate that the GH transgene initiated effects on growth enhancement in the early developmental stages and played an important role in improving protein synthesis and lipolysis. Conversely, homozygous transgenic fish No. 14960 grew faster than the control fish, but the culture traits of its hemizygous offspring (#TG1 line) were comparatively reduced, including lower SGR, FCE, PRE and ERE levels, and higher FR and lipid contents. Our results suggest that even if fast-growing GH-transgenic homozygous parental fish are generated, we still need to practice classical selective breeding in order to produce transgenic fish with improved traits that can meet aquaculture demand. (C) 2012 Elsevier B. V. All rights reserved.
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  • Differences in responses of grass carp to different types of grass carp reovirus (GCRV) and the mechanism of hemorrhage revealed by transcriptome sequencing

    He, Libo   Zhang, Aidi   Pei, Yongyan   Chu, Pengfei   Li, Yongming   Huang, Rong   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    Background: Grass carp is an important farmed fish in China that is affected by serious disease, especially hemorrhagic disease caused by grass carp reovirus (GCRV). The mechanism underlying the hemorrhagic symptoms in infected fish remains to be elucidated. Although GCRV can be divided into three distinct subtypes, differences in the pathogenesis and host immune responses to the different subtypes are still unclear. The aim of this study was to provide a comprehensive insight into the grass carp response to different GCRV subtypes and to elucidate the mechanism underlying the hemorrhagic symptoms. Results: Following infection of grass carp, GCRV-I was associated with a long latent period and low mortality (42.5%), while GCRV-II was associated with a short latent period and high mortality (81.4%). The relative copy number of GCRV-I remained consistent or decreased slightly throughout the first 7 days post-infection, whereas a marked increase in GCRV-II high copy number was detected at 5 days post-infection. Transcriptome sequencing revealed 211 differentially expressed genes (DEGs) in Group I (66 up-regulated, 145 down-regulated) and 670 (386 up-regulated, 284 down-regulated) in Group II. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment in the terms or pathways involved in immune responses and correlating with blood or platelets. Most of the DEGs in Group I were also present in Group II, although the expression profiles differed, with most DEGs showing mild changes in Group I, while marked changes were observed in Group II, especially the interferon-related genes. Many of the genes involved in the complement pathway and coagulation cascades were significantly up-regulated at 7 days post-infection in Group II, suggesting activation of these pathways. Conclusion: GCRV-I is associated with low virulence and a long latent period prior to the induction of a mild host immune response, whereas GCRV-II is associated with high virulence, a short latent period and stimulates a strong and extensive host immune response. The complement and coagulation pathways are significantly activated at 7 days post-infection, leading to the endothelial cell and blood cell damage that result in hemorrhagic symptoms.
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  • GABAergic Neurons and Their Modulatory Effects on GnRH3 in Zebrafish

    Song, Yanlong   Tao, Binbin   Chen, Ji   Jia, Shaoting   Zhu, Zuoyan   Trudeau, Vance L.   Hu, Wei  

    gamma-Aminobutyric acid (GABA) is a major amino acid neurotransmitter in the vertebrate brain. To provide detailed information on the distribution of the GABA in zebrafish (Danio rerio), neurons were labeled with mCherry driven by the glutamic acid decarboxylase 67 (gad67) promoter. In the transgenic line Tg(gad67: mCherry), mCherry-positive gad67 cell bodies were predominantly localized to the olfactory bulb, pallial zones, subpallium zones, parvocellular preoptic nucleus, periventricular gray zone of optic tectum, torus semicircularis, posterior tuberculum, medial longitudinal fascicle, caudal zone of periventricular hypothalamus, and oculomotor nucleus. mCherry-positive fibers were widely distributed in the olfactory bulbs, subpallium, thalamus, ventral hypothalamic zone, tectumopticum, mesencephalon, and rhombencephalon. mCherry-positive neurons were also observed in the retina and the spinal cord. The anatomical relationships between GABAergic and gonadotrophin-releasing hormone 3 (GnRH3) neurons were investigated by crossing Tg(gad67: mCherry) fish with the previously established Tg(gnrh3: EGFP) transgenic line. GnRH3 cell bodies and fibers were contacted by GABAergic fibers directly in the ventral telencephalon and anterior tuberal nucleus. A subpopulation of GnRH3 neurons in the ventral telencephalic area was also labeled with mCherry, so some GnRH3 neurons are also GABAergic. GABA(B) receptor agonist (baclofen) and antagonist (CGP55845) treatments indicated that GABA(B) receptor signaling inhibited gnrh3 expression in larval fish but was stimulatory in adult fish. The expression of pituitary lh beta and fsh beta was stimulated by intraperitoneal injection of baclofen in adult fish. We conclude that GABA via GABAB receptors regulates GnRH3 neurons in a developmentally dependent manner in zebrafish.
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  • Direct production of XY(DMY-) sex reversal female medaka (Oryzias latipes) by embryo microinjection of TALENs.

    Luo, Daji   Liu, Yun   Chen, Ji   Xia, Xiaoqin   Cao, Mengxi   Cheng, Bin   Wang, Xuejuan   Gong, Wuming   Qiu, Chao   Zhang, Yunsheng   Cheng, Christopher Hon Ki   Zhu, Zuoyan   Hu, Wei  

    Medaka is an ideal model for sex determination and sex reversal, such as XY phenotypically female patients in humans. Here, we assembled improved TALENs targeting the DMY gene and generated XY(DMY-) mutants to investigate gonadal dysgenesis in medaka. DMY-TALENs resulted in indel mutations at the targeted loci (46.8%). DMY-nanos3UTR-TALENs induced mutations were passed through the germline to F1 generation with efficiencies of up to 91.7%. XY(DMY-) mutants developed into females, laid eggs, and stably passed the Y(DMY-) chromosome to next generation. RNA-seq generated 157 million raw reads from WT male (WT_M_TE), WT female (WT_F_OV) and XY(DMY-) female medaka (TA_F_OV) gonad libraries. Differential expression analysis identified 144 up- and 293 down-regulated genes in TA_F_OV compared with WT_F_OV, 387 up- and 338 down-regulated genes in TA_F_OV compared with WT_M_TE. According to genes annotation and functional prediction, such as Wnt1 and PRCK, it revealed that incomplete ovarian function and reduced fertility of XY(DMY-) mutant is closely related to the wnt signaling pathway. Our results provided the transcriptional profiles of XY(DMY-) mutants, revealed the mechanism between sex reversal and DMY in medaka, and suggested that XY(DMY-) medaka was a novel mutant that is useful for investigating gonadal dysgenesis in phenotypic female patients with the 46, XY karyotype. =20
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  • Cloning and characterization of the LEF/TCF gene family in grass carp (Ctenopharyngodon idella) and their expression profiles in response to grass carp reovirus infection

    Zhu, Denghui   Huang, Rong   Chen, Liangming   Fu, Peipei   Luo, Lifei   He, Libo   Li, Yongming   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

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  • Growth enhancement of crucian carp (Carassius auratus gibelio Bloch) from human growth hormone gene transfer and human growth hormone administration

    Xu, Keshang   Wei, Yanzhang   Guo, Lihe   Zhu, Zuoyan  

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  • The draft genome of the grass carp (Ctenopharyngodon idellus) provides insights into its evolution and vegetarian adaptation.

    Wang, Yaping   Lu, Ying   Zhang, Yong   Ning, Zemin   Li, Yan   Zhao, Qiang   Lu, Hengyun   Huang, Rong   Xia, Xiaoqin   Feng, Qi   Liang, Xufang   Liu, Kunyan   Zhang, Lei   Lu, Tingting   Huang, Tao   Fan, Danlin   Weng, Qijun   Zhu, Chuanrang   Lu, Yiqi   Li, Wenjun   Wen, Ziruo   Zhou, Congcong   Tian, Qilin   Kang, Xiaojun   Shi, Mijuan   Zhang, Wanting   Jang, Songhun   Du, Fukuan   He, Shan   Liao, Lanjie   Li, Yongming   Gui, Bin   He, Huihui   Ning, Zhen   Yang, Cheng   He, Libo   Luo, Lifei   Yang, Rui   Luo, Qiong   Liu, Xiaochun   Li, Shuisheng   Huang, Wen   Xiao, Ling   Lin, Haoran   Han, Bin   Zhu, Zuoyan  

    The grass carp is an important farmed fish, accounting for 16% of global freshwater aquaculture, and has a vegetarian diet. Here we report a 0.9-Gb draft genome of a gynogenetic female adult and a 1.07-Gb genome of a wild male adult. Genome annotation identified 27,263 protein-coding gene models in the female genome. A total of 114 scaffolds consisting of 573 Mb are anchored on 24 linkage groups. Divergence between grass carp and zebrafish is estimated to have occurred 49-54 million years ago. We identify a chromosome fusion in grass carp relative to zebrafish and report frequent crossovers between the grass carp X and Y chromosomes. We find that transcriptional activation of the mevalonate pathway and steroid biosynthesis in liver is associated with the grass carp's adaptation from a carnivorous to an herbivorous diet. We believe that the grass carp genome could serve as an initial platform for breeding better-quality fish using a genomic approach. =20
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  • Transcriptome analysis of head kidney in grass carp and discovery of immune-related genes

    Chen, Jin   Li, Cai   Huang, Rong   Du, Fukuan   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

    Background: Grass carp (Ctenopharyngodon idella) is one of the most economically important freshwater fish, but its production is often affected by diseases that cause serious economic losses. To date, no good breeding varieties have been obtained using the oriented cultivation technique. The ability to identify disease resistance genes in grass carp is important to cultivate disease-resistant varieties of grass carp. Results: In this study, we constructed a non-normalized cDNA library of head kidney in grass carp, and, after clustering and assembly, we obtained 3,027 high-quality unigenes. Solexa sequencing was used to generate sequence tags from the transcriptomes of the head kidney in grass carp before and after grass carp reovirus (GCRV) infection. After processing, we obtained 22,144 tags that were differentially expressed by more than 2-fold between the uninfected and infected groups. 679 of the differentially expressed tags (3.1%) mapped to 483 of the unigenes (16.0%). The up-regulated and down-regulated unigenes were annotated using gene ontology terms; 16 were annotated as immune-related and 42 were of unknown function having no matches to any of the sequences in the databases that were used in the similarity searches. Semi-quantitative RT-PCR revealed four unknown unigenes that showed significant responses to the viral infection. Based on domain structure predictions, one of these sequences was found to encode a protein that contained two transmembrane domains and, therefore, may be a transmembrane protein. Here, we proposed that this novel unigene may encode a virus receptor or a protein that mediates the immune signalling pathway at the cell surface. Conclusion: This study enriches the molecular basis data of grass carp and further confirms that, based on fish tissue-specific EST databases, transcriptome analysis is an effective route to discover novel functional genes.
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  • Identification and molecular characterization of peroxiredoxin 2 in grass carp (Ctenopharyngodon idella)

    Zhu, Denghui   Huang, Rong   Yang, Cheng   Fu, Peipei   Chen, Liangming   Jiang, Yinjun   He, Libo   Li, Yongming   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

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  • Molecular characterization and functional activity of Prx1 in grass carp (Ctenopharyngodon idella)

    Zhu, Denghui   Li, Yangyang   Huang, Rong   Luo, Lifei   Chen, Liangming   Fu, Peipei   He, Libo   Li, Yongming   Liao, Lanjie   Zhu, Zuoyan   Wang, Yaping  

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  • Expression of foreign genes during embryogenesis

    Zou, Jun   Xie, Yuefeng   Liu, Dong   Zhu, Zuoyan  

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  • Fam60al as a novel factor involved in reprogramming of somatic cell nuclear transfer in zebrafish (Danio rerio).

    Hu, Hongling   Tao, Binbin   Chen, Ji   Zhu, Zuoyan   Hu, Wei  

    The main reason for abnormal development of cloned animals or embryos, and inefficient animal cloning, is a poor understanding of the reprogramming mechanism. To better comprehend reprogramming and subsequent generation of pluripotent stem cells, we must investigate factors related to reprogramming of somatic cells as nuclear donors. As we know, fam60al (family with sequence similarity 60, member A, like) is a coding gene only found in zebrafish and frog (Xenopus laevis) among vertebrates. However, until now, its functions have remained unknown. Here, we generated a zebrafish fam60al-/- mutant line using transcription activator-like effector nucleases (TALENs), and found that both nanog and klf4b expression significantly decreased while myca expression significantly increased in fam60al-/- mutant embryos. Concurrently, we also uncovered that in developmentally arrested embryos of somatic cell nuclear transfer, nanog, klf4b and myca expression was down-regulated, accompanying a decrease of fam60al expression. Interestingly, we identified a long noncoding RNA (lncRNA) of fam60al, named fam60al-AS, which negatively regulated fam60al by forming double-stranded RNA (dsRNA). RNase protection assay and real-time PCR confirmed these findings. Taken together, these results suggest that fam60al is a novel factor related to the reprogramming of somatic cell nuclear transfer in zebrafish, which is regulated by its reverse lncRNA.=20
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  • Heritable/conditional genome editing in C. elegans using a CRISPR-Cas9 feeding system

    Liu, Pengpeng   Long, Lijiang   Xiong, Kai   Yu, Bo   Chang, Nannan   Xiong, Jing-Wei   Zhu, Zuoyan  

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  • Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection.

    Su, Jianguo   Zhu, Zuoyan   Wang, Yaping   Zou, Jun   Hu, Wei  

    Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P < 0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.
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