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Zhiyuan Gong - One of the best experts on this subject based on the ideXlab platform.
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lipopolysaccharides enhance epithelial hyperplasia and tubular adenoma in intestine specific expression of krasv12 in Transgenic Zebrafish
Biomedicines, 2021Co-Authors: Yuxi Sun, Pei Shi Angelina Fong, Liangin Lin, Dong Liu, Zhiyuan GongAbstract:Intestinal carcinogenesis is a multistep process that begins with epithelial hyperplasia, followed by a transition to an adenoma and then to a carcinoma. Many etiological factors, including KRAS mutations and inflammation, have been implicated in oncogenesis. However, the potential synergistic effects between KRAS mutations and inflammation as well as the potential mechanisms by which they promote intestinal carcinogenesis remain unclear. Thus, the objective of this study was to investigate the synergistic effects of krasV12, lipopolysaccharides (LPS), and/or dextran sulfate sodium (DSS) on inflammation, tumor progression, and intestinal disorders using Transgenic adults and larvae of Zebrafish. Histopathology and pathological staining were used to examine the intestines of krasV12 Transgenic Zebrafish treated with LPS and/or DSS. LPS and/or DSS treatment enhanced intestinal inflammation in krasV12 Transgenic larvae with concomitant increases in the number of neutrophils and macrophages in the intestines. The expression of krasV12, combined with LPS treatment, also enhanced epithelial hyperplasia and tubular adenoma, demonstrated by histopathological examinations and by increases in cell apoptosis, cell proliferation, and downstream signaling of phosphorylated AKT serine/threonine kinase 1 (AKT), extracellular-signal-regulated kinase (ERK), and histone. We also found that krasV12 expression, combined with LPS treatment, significantly enhanced changes in intestinal morphology, specifically (1) decreases in goblet cell number, goblet cell size, villi height, and intervilli space, as well as (2) increases in villi width and smooth muscle thickness. Moreover, krasV12 Transgenic larvae cotreated with DSS and LPS exhibited exacerbated intestinal inflammation. Cotreatment with DSS and LPS in krasV12-expressing Transgenic adult Zebrafish also enhanced epithelial hyperplasia and tubular adenoma, compared with wild-type fish that received the same cotreatment. In conclusion, our data suggest that krasV12 expression, combined with LPS and/or DSS treatment, can enhance intestinal tumor progression by activating the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway and may provide a valuable in vivo platform to investigate tumor initiation and antitumor drugs for gastrointestinal cancers.
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Exacerbation of Liver Tumor Metastasis in twist1a+/xmrk+ Double Transgenic Zebrafish following Lipopolysaccharide or Dextran Sulphate Sodium Exposure
'MDPI AG', 2021Co-Authors: Yuxi Sun, Liangin Lin, Dong Liu, Zhiyuan GongAbstract:The poor prognosis for patients with hepatocellular carcinoma (HCC) is related directly to metastasis. The Twist1 gene encodes for a transcription factor essential to embryogenesis. It has also been shown to promote epithelial-to-mesenchymal transition (EMT), invasion, and metastasis; however, there is currently no in vivo evidence that Twist1 plays a role in the metastasis of liver tumors. Zebrafish are increasingly being used as an alternative cancer model. In the current study, an adult-stage Zebrafish HCC model was used to examine the synergistic effects of twist1a and xmrk, a well characterized oncogene, during HCC metastasis. We also examined the effects of two inflammatory agents, lipopolysaccharides (LPS) and dextran sulfate sodium (DSS), on the hepatocyte-specific expression of Transgenic twist1a and xmrk. The conditional overexpression of twist1a and xmrk was shown to promote liver tumor metastasis in Zebrafish, resulting in increased apoptosis and cell proliferation as well as tumor maintenance and propagation independent of the inherent EMT-inducing activity of xmrk. Exposing twist1a+/xmrk+ Transgenic Zebrafish to LPS or DSS was shown to promote metastasis, indicating that the overexpression of twist1a and xmrk led to crosstalk between the signaling pathways involved in EMT. This study provides important evidence pertaining to the largely overlooked effects of signaling crosstalk between twist1a and xmrk in regulating HCC metastasis. Our results also suggest that the co-expression of twist1a/xmrk in conjunction with exposure to LPS or DSS enhances HCC metastasis, and provides a valuable in vivo platform by which to investigate tumor initiation and metastasis in the study of liver cancer
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transcriptomic analyses of oncogenic hepatocytes reveal common and different molecular pathways of hepatocarcinogenesis in different developmental stages and genders in kras g12v Transgenic Zebrafish
Biochemical and Biophysical Research Communications, 2019Co-Authors: Xiaojing Huo, Chuan Yan, Sinnakaruppan Mathavan, Jianjun Liu, Zhiyuan GongAbstract:Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is mainly due to genetic changes in hepatocytes. However, molecular expression in hepatocytes during hepatocarcinogenesis has not been characterized. In this study, using an inducible kras Transgenic Zebrafish models for HCC, transcriptomic profiles of oncogenic hepatocytes from larvae, male and female adult fish following a brief induction of oncogenic kras were investigated. We found that oncogenic hepatocytes from all the three sources possess most of the cancer hallmarks at molecular level, including Sustaining proliferative signaling, Evading growth suppressors, Resisting cell death, Avoiding immune destruction, Inflammation, Reprogramming of energy metabolism, Angiogenesis, and Activating invasion and metastasis, suggesting the malignant transformation at molecular level could occur at the early stage of hepatocarcinogensis and can be captured in hepatocytes. However, each group of oncogenic hepatocytes also had their own characteristics. Larval oncogenic hepatocytes have cancer stem cell features. Female oncogenic hepatocytes showed resemblance to a mild human HCC subtype while male oncogenic hepatocytes resembled a severe HCC subtype, consistent with the observed sex disparity of HCC in both Zebrafish and human. Finally, the two adult groups were more similar to each other than to the larval group, indicating an overwhelming effect of development over the gender.
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Inducible Intestine-Specific Expression of krasV12 Triggers Intestinal Tumorigenesis In Transgenic Zebrafish
Elsevier, 2018Co-Authors: Divya Raghuram, Pei Shi Angelina Fong, Zhiyuan GongAbstract:KRAS mutations are a major risk factor in colorectal cancers. In particular, a point mutation of KRAS of amino acid 12, such as KRAS V12, renders it stable activity in oncogenesis. We found that krasV12 promotes intestinal carcinogenesis by generating a Transgenic Zebrafish line with inducible krasV12 expression in the intestine, Tg(ifabp:EGFP-krasV12). The Transgenic fish generated exhibited significant increases in the rates of intestinal epithelial outgrowth, proliferation, and cross talk in the active Ras signaling pathway involving in epithelial-mesenchymal transition (EMT). These results provide in vivo evidence of Ras pathway activation via krasV12 overexpression. Long-term Transgenic expression of krasV12 resulted in enteritis, epithelial hyperplasia, and tubular adenoma in adult fish. This was accompanied by increased levels of the signaling proteins p-Erk and p-Akt and by downregulation of the EMT marker E-cadherin. Furthermore, we also observed a synergistic effect of krasV12 expression and dextran sodium sulfate treatment to enhance intestinal tumor in Zebrafish. Our results demonstrate that krasV12 overexpression induces intestinal tumorigenesis in Zebrafish, which mimics intestinal tumor formation in humans. Thus, our Transgenic Zebrafish may provide a valuable in vivo platform that can be used to investigate tumor initiation and anticancer drugs for gastrointestinal cancers
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generation of tg cyp1a gfp Transgenic Zebrafish for development of a convenient and sensitive in vivo assay for aryl hydrocarbon receptor activity
Marine Biotechnology, 2015Co-Authors: Xiaoyan Zhang, Chunsheng Liu, Zhiyuan GongAbstract:Both dioxins/dioxin-like compounds and polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants and cause multiple adverse health effects on human and wildlife. Cyp1a is the most commonly used biomarker induced by these pollutants through activation of the aryl hydrocarbon receptor (AhR) pathway. Here we generated Tg(cyp1a:gfp) Transgenic Zebrafish for establishing a convenient in vivo assay for analysing these xenobiotic compounds. The Tg(cyp1a:gfp) larvae at 4 day post-fertilization were tested with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and GFP induction was observed mainly in the kidney, liver and gut. Similar GFP expression was also induced strongly by two dioxin-like chemicals, co-planar polychlorinated biphenyl (PCB126) and polychlorinated dibenzo-p-furan (PeCDF) and relatively weakly by two PAHs, 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BAP). The lowest observed effective concentration (LOEC) of TCDD was estimated to be ∼1 pM and the EC50 (effective concentration to induce GFP in 50 % of Tg(cyp1a:gfp) larvae) was ∼10 pM. PCB126 and PeCDF had ∼10× lower potencies in GFP induction than TCDD, while the potencies for 3-MC and BAP were at least 1000× lower. The sensitivity of Tg(cyp1a:gfp) larvae to respond TCDD was also favourable compared to that of ethoxyresorufin-O-deethylase (EROD) assay in both Zebrafish larvae and adult livers. As GFP-based assay in Transgenic Zebrafish can be easily accommodated in multi-well dishes, the Tg(cyp1a:gfp) Zebrafish should provide not only a valuable biomonitoring tool for aquatic contaminants but also a potential high-throughput chemical screening platform for identification of new AhR agonists.
Thomas A Look - One of the best experts on this subject based on the ideXlab platform.
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epigenetic therapy inhibits nup98 hoxa9 mediated myeloid disease decitabine and valproic acid work synergistically to rescue normal hematopoiesis in Transgenic Zebrafish
Blood, 2012Co-Authors: Adam P Deveau, Michael A Forrester, Clemens Grabher, Thomas A Look, Andrew J Coombs, Ian C Chute, Daniel Leger, Stephen M Lewis, Jason N BermanAbstract:Abstract 2391 Acute myeloid leukemia (AML) results from multiple genetic lesions that alter white blood cell development, leading to hyperproliferation and a block in differentiation. A better understanding of these molecular pathways will enable the development of therapies that selectively target the specific abnormality in a leukemic cell with improved outcome and limited toxicity. Pertinent animal models serve as essential intermediaries between in vitro studies and the use of these new agents in clinical trials. The NUP98-HOXA9 ( NHA9 ) fusion oncogene is found in high risk de novo AML, treatment related AML and chronic myeloid leukemia (CML) blast crisis. We previously generated a Transgenic Zebrafish model overexpressing human NHA 9 under the Zebrafish pu.1 promoter (Forrester et al, BJH , 2012). Almost 25% of adult NHA9 -Transgenic fish develop a myeloproliferative neoplasm (MPN) in the kidney marrow, the site of adult hematopoiesis. Additionally, NHA9- Transgenic embryos demonstrate an increase in immature myeloid cells ( l-plastin1 , 2.3-fold by in situ , P gata1 , 19.3-fold by qRT-PCR, P NHA9 Zebrafish provides an unprecedented opportunity to perform in vivo screens for collaborating genes in NHA9 -induced leukemogenesis and evaluate the impact of molecularly targeted agents. Microarray analysis found high expression of DNA (cytosine-5-)-methyltransferase 1 ( dnmt1 ) in NHA9 embryos, which was confirmed by qRT-PCR (3.4-fold increase). The methylating activity of the DNMT1 enzyme is part of the epigenetic machinery that represses genes needed for terminal myeloid differentiation. Overexpression of human DNMT1 has been found in some cases of AML, but has not been previously linked with NHA9 -induced disease. The overexpression of the Zebrafish dnmt1 homolog in NHA9 embryos may keep cells trapped in an immature state, a hallmark of AML. Injecting NHA9 embryos with a gene-blocking dnmt1 morpholino restored normal hematopoiesis with wild-type expression levels of both l-plastin and gata1 . Similarly, a dose-dependent return to normal proportions of myeloid and erythroid cells was achieved by bathing NHA9 embryos in 50–100 μM decitabine (5-aza-29-deoxycytidine), a demethylating agent that specifically targets the DNMT1 enzyme. However, decitabine use as a monotherapy carries the risk of genomic instability due to wide-spread DNA hypomethylation. We therefore considered treating our NHA9 Zebrafish embryos with combination therapy against multiple molecular targets. DNMT1 is part of a larger epigenetic machinery and works in parallel with histone deacetylation complexes (HDACs). Similar to decitabine, exposing NHA9 embryos to 150–250 μM of the HDAC inhibitor, valproic acid (VPA), lead to a dose-dependent return of normal hematopoiesis. In vitro studies suggest that combined treatment with a demethylating agent and an HDAC inhibitor may synergize to be more effective than either compound alone in combating myeloid disease, permitting use of lower drug doses, thus avoiding unnecessary toxicity. Indeed, we found that combination doses as low as 10 μM each of decitabine and VPA restored normal hematopoiesis as effectively as significantly higher doses of each monotherapy. This drug synergy identified in our Transgenic Zebrafish directly links NHA9 -induced leukemia and epigenetic regulation and has set the stage for a new and exciting therapeutic approach for high risk AML. True to our goal, translating this treatment to clinical studies may ultimately improve outcome and minimize toxic side-effects, thereby increasing the long-term survival of patients with high-risk AML. Disclosures: No relevant conflicts of interest to declare.
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nup98 hoxa9 reprograms embryonic hematopoiesis suppresses cellular apoptosis and causes malignant tissue infiltrates in Transgenic Zebrafish
Blood, 2009Co-Authors: Michael A Forrester, Clemens Grabher, Eileen R Mcbride, Ellen R Boyd, Fuiboon Kai, Sahar I Daas, Thomas A Look, Jason N BermanAbstract:Abstract 3961 Poster Board III-897 The NUP98-HOXA9 fusion oncogene results from a t(7;11)(p15;p15) chromosomal translocation and is associated with inferior prognosis in de novo and treatment-related acute myeloid leukemia (AML), as well as blast crisis in chronic myeloid leukemia (CML). HOXA9 belongs to the highly-conserved HOX gene family of developmental transcription factors and is critical for vertebrate hematopoiesis. Upregulation of HOXA9 is a frequent phenomenon in human AML, implicating a central role in myeloid disease. Thus, elucidating the activity of oncogenic NUP98-HOXA9 may reveal universal mechanisms of leukemogenesis and lead to design of targeted therapies. The Zebrafish is a robust model for studying vertebrate hematopoiesis and leukemogenesis, by virtue of its ex utero development, and conserved genetics and cell biology. We have engineered a Cre- lox inducible Transgenic Zebrafish harboring the human NUP98-HOXA9 fusion oncogene downstream of the Zebrafish pu.1 promoter. NUP98-HOXA9 perturbed Zebrafish embryonic hematopoiesis, leading to upregulated expression of myeloid-specific genes, pu.1 , lysC , and l-plastin , and downregulation of the erythroid-specific gene, gata1 . These changes in gene transcription affect both ‘primitive’ and ‘definitive’ waves of Zebrafish hematopoiesis (including the recently-described erythro-myeloid progenitors [EMPs]), suggesting that NUP98-HOXA9 reprograms blood cell precursors to a predominantly myeloid fate. Preferential upregulation of pu.1 during ‘primitive’ hematopoiesis further suggests an impaired potential for terminal myeloid differentiation. These effects appear developmentally mediated, as cell proliferation was not increased as measured by phosphorylated histone-H3 (pH3) staining. However, following ionizing irradiation (IR), NUP98-HOXA9 expression preserved cell proliferation and suppressed apoptosis in the presence of DNA damage. Irradiated Transgenic embryos displayed wild-type levels of pH3, suggesting inhibition of cell cycle checkpoints. Irradiated embryos also displayed reduced levels of the conserved apoptotic marker, activated caspase-3, as well as downregulation of the puma pro-apoptotic gene, and upregulation of bcl2 and bcl-xL anti-apoptotic genes. These data postulate suppression of caspase-3-dependent apoptosis as a mechanism for NUP98-HOXA9 -mediated oncogenesis in vivo . Incomplete phenotypic ‘rescue’ with bcl-xL morpholino suggests insufficiency of single-target therapy, with a need for multi-target interruptions to restore radiosensitivity. Between 20-24 months post fertilization, a proportion of these NUP98-HOXA9 Transgenic Zebrafish (n=9/19; 47%) presented with gross abdominal masses and/or abnormal swimming behavior. Thus far, histological sectioning has demonstrated enlarged kidneys and livers, with evidence of infiltrative, pleiomorphic, mitotically-active cells with myeloid morphology. This pathology is reminiscent of the myeloproliferative disease (MPD) observed in NUP98-HOXA9 Transgenic mice. Taken together, these data provide insights into the mechanisms underlying NUP98-HOXA9 -mediated leukemogenesis and provide an unprecedented opportunity for in vivo chemical modifier screens to identify promising therapeutic agents that restore a normal phenotype. Disclosures: No relevant conflicts of interest to declare.
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oncogenic hoxa9 inhibits cellular apoptosis induced by ionizing radiation ir in Transgenic Zebrafish
Blood, 2008Co-Authors: Ellen R Boyd, Michael A Forrester, Clemens Grabher, Fuiboon Kai, Sahar I Daas, Thomas A Look, Jason N BermanAbstract:Acute myeloid leukemia (AML) is characterized by the failure of terminal cellular differentiation of the myeloid lineage, and retains a significant rate of mortality despite aggressive therapy. One key oncogenic candidate, HOXA9 – belonging to the highly-conserved HOX family of developmental transcription factors – is frequently overexpressed in AML. In particular, this may result from the rare, but recurrent t(7;11)(p15;p15) chromosomal translocation, yielding the chimeric transcription factor NUP98-HOXA9 . This translocation event is associated with a poor prognosis and elucidating the molecular activity of this fusion oncogene will enable the development of targeted therapeutics. Despite established mammalian models of AML, specific transcriptional targets of HOXA9 remain poorly defined. The Zebrafish, Danio rerio , possesses orthologous blood cells to humans, making it a reliable model of vertebrate hematopoiesis and a promising tool for studying the molecular mechanisms underlying AML. The Zebrafish has many advantages over traditional mouse models: chiefly, the ex utero development and transparency of Zebrafish embryos allow for ease of manipulation, phenotypic analysis, and high throughput screening. We have demonstrated that ubiquitous overexpression of the human NUP98-HOXA9 oncogene in germline Transgenic Zebrafish embryos protects against apoptosis induced by ionizing radiation (IR) – a hallmark of oncogenesis. Acridine orange staining was indicative of decreased apoptosis in live Transgenic embryos compared with wild type (WT) controls. Using whole mount immunofluorescent labeling, we similarly demonstrated that IR-treated Transgenic embryos have a significant reduction in levels of activated caspase-3, a downstream effector protein in the conserved caspase cascade. By comparison, only a mild reduction in IR-induced apoptosis and activated caspase-3 was observed in a second Transgenic Zebrafish line overexpressing murine native Hoxa9 , suggesting a gradient of impact on cell survival. Taken together, these findings implicate a block in caspase-3-dependent apoptosis as a mechanism underlying HOXA9 -mediated oncogenesis in vivo and provide an opportunity to perform chemical modifier screens to identify agents that restore a WT phenotype. Whole mount RNA in situ hybridization for expression of pro-apoptotic genes, bad and puma , and anti-apoptotic genes, bcl2 and bcl-x L , will provide further insight into the effects of oncogenic HOXA9 on the caspase cascade. Similarly, rescue of caspase-3 activation and IR-induced apoptosis is underway via microinjection of bad and puma mRNA. To achieve robust and continuous myeloid-specific expression of oncogenic HOXA9 and partners, new Transgenic strategies using both the Tol2 transposon-based Gateway cloning system and Cre/lox-induction are being employed. These lines will enable evaluation of myeloid-specific apoptosis, cell cycle regulation and cellular maturation to corroborate our findings in the ubiquitously-expressing lines, providing further insight into leukemogenic mechanisms and the potential to identify novel targeted therapies to improve the outcome in human AML.
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heat shock induction of t cell lymphoma leukaemia in conditional cre lox regulated Transgenic Zebrafish
British Journal of Haematology, 2007Co-Authors: Hui Feng, John P. Kanki, David M Langenau, Jennifer A Madge, Andre Quinkertz, Alejandro Gutierrez, Donna Neuberg, Thomas A LookAbstract:Summary The Zebrafish is an ideal vertebrate model system to investigate the complex genetic basis of cancer because it has the capacity for in vivo tumour-cell imaging and forward genetic screens, and the molecular mechanisms regulating malignancy are remarkably conserved when compared with human. Our laboratory has previously generated Transgenic Zebrafish models that overexpress the mouse c-Myc gene fused to enhanced green fluorescent protein (EGFP) and develop T-cell acute lymphoblastic leukaemia (T-ALL) that recapitulates the human disease both molecularly and pathologically. Our previous models have been limited by disease onset prior to sexual maturity and by the low disease penetrance when conditional Transgenic embryos are injected with Cre RNA. Here, we report a novel system in which compound Transgenic fish expressed both Cre controlled by the heat-shock promoter and a rag2-promoter-regulated lox-dsRED2-lox-EGFP-mMyc cassette rag2-LDL-EMyc in developing T cells. After heat-shock treatment at 3 d postfertilisation (dpf) for 45 min at 37°C, 81% of compound Transgenic fish developed T-lymphoblastic lymphoma (T-LBL, mean latency 120 ± 43 (standard deviation) days of life), which rapidly progressed to T-ALL. Heat-shock-regulated Transgenic technology in Zebrafish provides the missing link necessary to exploit the powerful genetic capacity of this organism to probe the multi-step molecular pathogenesis of leukaemia.
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resolution of inflammation by retrograde chemotaxis of neutrophils in Transgenic Zebrafish
Journal of Leukocyte Biology, 2006Co-Authors: Jonathan R Mathias, John P. Kanki, Thomas A Look, Benjamin J Perrin, Ting Xi Liu, Anna HuttenlocherAbstract:Neutrophil chemotaxis to sites of inflammation is a critical process during normal immune responses to tissue injury and infection and pathological immune responses leading to chronic inflammation. Although progress has been made in understanding the mechanisms that promote neutrophil recruitment to inflamed tissue, the mechanisms that regulate the resolution phase of the inflammatory response have remained relatively elusive. To define the mechanisms that regulate neutrophil-mediated inflammation in vivo, we have developed a novel Transgenic Zebrafish in which the neutrophils express GFP under control of the myeloperoxidase promoter (zMPO:GFP). Tissue injury induces a robust, inflammatory response, which is characterized by the rapid chemotaxis of neutrophils to the wound site. In vivo time-lapse imaging shows that neutrophils subsequently display directed retrograde chemotaxis back toward the vasculature. These findings implicate retrograde chemotaxis as a novel mechanism that regulates the resolution phase of the inflammatory response. The zMPO:GFP Zebrafish provides unique insight into the mechanisms of neutrophil-mediated inflammation and thereby offers opportunities to identify new regulators of the inflammatory response in vivo.
Zuoyan Zhu - One of the best experts on this subject based on the ideXlab platform.
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vitreoscilla hemoglobin vhb overexpression increases hypoxia tolerance in Zebrafish danio rerio
Marine Biotechnology, 2011Co-Authors: Bo Guan, Yaping Wang, Zhongping Lin, Zuoyan ZhuAbstract:Aquaculture farming may benefit from genetically engineering fish to tolerate environmental stress. Here, we used the vector pCVCG expressing the Vitreoscilla hemoglobin (vhb) gene driven by the common carp β-actin promoter to create stable Transgenic Zebrafish. The survival rate of the 7-day-old F2 Transgenic fish was significantly greater than that of the sibling controls under 2.5% O2 (dissolved oxygen (DO), 0.91 mg/l). Meanwhile, we investigated the relative expression levels of several marker genes (hypoxia-inducible factor alpha 1, heat shock cognate 70-kDa protein, erythropoietin, beta and alpha globin genes, lactate dehydrogenase, catalase, superoxide dismutase, and glutathione peroxidase) of Transgenic fish and siblings after hypoxia exposure for 156 h. The expression profiles of the vhb Transgenic Zebrafish revealed that VHb could partially alleviate the hypoxia stress response to improve the survival rate of the fish. These results suggest that that vhb gene may be an efficient candidate for genetically modifying hypoxia tolerance in fish.
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non homologous end joining plays a key role in transgene concatemer formation in Transgenic Zebrafish embryos
International Journal of Biological Sciences, 2010Co-Authors: Jun Dai, Xiaojuan Cui, Zuoyan ZhuAbstract:This study focused on concatemer formation and integration pattern of transgenes in Zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5'-protruding, 3'-protruding, and blunt ends that were microinjected into Zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or re moved by cutting. Repair of DSBs with symmetric 3'-3' protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of Transgenic Zebrafish.
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visualization of monoaminergic neurons and neurotoxicity of mptp in live Transgenic Zebrafish
Developmental Biology, 2008Co-Authors: Lu Wen, Shuo Lin, Zuoyan Zhu, Wei Wei, Peng Huang, Xi Ren, Zheng Zhang, Bo ZhangAbstract:We describe an enhancer trap Transgenic Zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 (vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinson's disease.
Jian Gao - One of the best experts on this subject based on the ideXlab platform.
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fat 1 Transgenic Zebrafish are protected from abnormal lipid deposition induced by high vegetable oil feeding
Applied Microbiology and Biotechnology, 2020Co-Authors: Shouxiang Sun, Filipe Castro, Oscar Monroig, Xiaojuan Cao, Jian GaoAbstract:High dietary concentration of vegetable oil, particularly those rich in n-6 polyunsaturated fatty acids (PUFAs), can induce negative physiological effects including excessive lipid deposition in teleost fish. Omega-3 desaturase (Fat-1) of Caenorhabditis elegans is able to convert n-6 PUFAs to n-3 PUFAs and thus induces a low n-6/n-3 PUFAs ratio alleviating lipid deposition. In this study, we investigated the effects of dietary n-6 PUFAs on lipid metabolism of fat-1 Transgenic Zebrafish (Tg:fat-1), to explore the role of fat-1 in fish lipid metabolism. We first generated Tg:fat-1 Zebrafish and assayed the effects of a low-fat diet (LFD) and a high-fat diet (HFD) prepared from soybean oil. Wild type Zebrafish (WT) fed with HFD (HFD-WT) exhibited increased obesity and lipid deposition, especially in the abdominal cavity and liver. These defects were absent from HFD-Tg:fat-1. For each diet group, Tg:fat-1 exhibited significantly decreased levels of almost all hepatic lipid classes compared with WT. Expression levels of lipid synthesis-related genes and lipid deposition-related genes were markedly lower in the liver of HFD-Tg:fat-1 compared with HFD-WT. In contrast, the steatolysis-related genes significantly upregulated in HFD-Tg:fat-1. Then expression profiles of mitochondrial energy metabolism-related genes and ATP contents in the livers from LFD-WT, LFD-Tg:fat-1, HFD-WT, and HFD-Tg:fat-1 were determined. Our findings suggest that fat-1 protects fish from abnormal lipid deposition induced by high-vegetable oil feeding, through endogenously converting n-6 PUFAs to n-3 PUFAs. KEY POINTS: • fat-1 Transgenic Zebrafish (Tg:fat-1) can endogenously convert n-6 PUFAs to n-3 PUFAs. • Tg:fat-1 avoid serious abnormal lipid deposition induced by high-vegetable oil feeding. • fat-1 transgenosis effectively improved lipid metabolism and mitochondrial energy metabolism in Zebrafish.
Bo Guan - One of the best experts on this subject based on the ideXlab platform.
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vitreoscilla hemoglobin vhb overexpression increases hypoxia tolerance in Zebrafish danio rerio
Marine Biotechnology, 2011Co-Authors: Bo Guan, Yaping Wang, Zhongping Lin, Zuoyan ZhuAbstract:Aquaculture farming may benefit from genetically engineering fish to tolerate environmental stress. Here, we used the vector pCVCG expressing the Vitreoscilla hemoglobin (vhb) gene driven by the common carp β-actin promoter to create stable Transgenic Zebrafish. The survival rate of the 7-day-old F2 Transgenic fish was significantly greater than that of the sibling controls under 2.5% O2 (dissolved oxygen (DO), 0.91 mg/l). Meanwhile, we investigated the relative expression levels of several marker genes (hypoxia-inducible factor alpha 1, heat shock cognate 70-kDa protein, erythropoietin, beta and alpha globin genes, lactate dehydrogenase, catalase, superoxide dismutase, and glutathione peroxidase) of Transgenic fish and siblings after hypoxia exposure for 156 h. The expression profiles of the vhb Transgenic Zebrafish revealed that VHb could partially alleviate the hypoxia stress response to improve the survival rate of the fish. These results suggest that that vhb gene may be an efficient candidate for genetically modifying hypoxia tolerance in fish.
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Vitreoscilla Hemoglobin (VHb) Overexpression Increases Hypoxia Tolerance in Zebrafish (Danio rerio)
2011Co-Authors: Bo Guan, Hu Wei, Wang Yaping, Zhu Zuoyan, Ma Hong, Hu Yuanlei, Lin Zhongping, Hu, W, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol Biotechnol, Wuhan 430072, Peo ChinaAbstract:Aquaculture farming may benefit from genetically engineering fish to tolerate environmental stress. Here, we used the vector pCVCG expressing the Vitreoscilla hemoglobin (vhb) gene driven by the common carp beta-actin promoter to create stable Transgenic Zebrafish. The survival rate of the 7-day-old F-2 Transgenic fish was significantly greater than that of the sibling controls under 2.5% O-2 (dissolved oxygen (DO), 0.91 mg/l). Meanwhile, we investigated the relative expression levels of several marker genes (hypoxia-inducible factor alpha 1, heat shock cognate 70-kDa protein, erythropoietin, beta and alpha globin genes, lactate dehydrogenase, catalase, superoxide dismutase, and glutathione peroxidase) of Transgenic fish and siblings after hypoxia exposure for 156 h. The expression profiles of the vhb Transgenic Zebrafish revealed that VHb could partially alleviate the hypoxia stress response to improve the survival rate of the fish. These results suggest that that vhb gene may be an efficient candidate for genetically modifying hypoxia tolerance in fish
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Vitreoscilla Hemoglobin (VHb) Overexpression Increases Hypoxia Tolerance in Zebrafish (Danio rerio)
marine biotechnology, 2011Co-Authors: Bo Guan, Wang Yaping, Zhu Zuoyan, Ma Hong, Hu Yuanlei, Lin Zhongping, Hu WeiAbstract:Aquaculture farming may benefit from genetically engineering fish to tolerate environmental stress. Here, we used the vector pCVCG expressing the Vitreoscilla hemoglobin (vhb) gene driven by the common carp beta-actin promoter to create stable Transgenic Zebrafish. The survival rate of the 7-day-old F(2) Transgenic fish was significantly greater than that of the sibling controls under 2.5% O(2) (dissolved oxygen (DO), 0.91 mg/l). Meanwhile, we investigated the relative expression levels of several marker genes (hypoxia-inducible factor alpha 1, heat shock cognate 70-kDa protein, erythropoietin, beta and alpha globin genes, lactate dehydrogenase, catalase, superoxide dismutase, and glutathione peroxidase) of Transgenic fish and siblings after hypoxia exposure for 156 h. The expression profiles of the vhb Transgenic Zebrafish revealed that VHb could partially alleviate the hypoxia stress response to improve the survival rate of the fish. These results suggest that that vhb gene may be an efficient candidate for genetically modifying hypoxia tolerance in fish.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000290039300024&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Biotechnology & Applied MicrobiologyMarine & Freshwater BiologySCI(E)EI13ARTICLE2336-3441
