Publications by authors named "Bensheng Ju"

22 Publications

  • Page 1 of 1

The miR-424(322)/503 gene cluster regulates pro- versus anti-inflammatory skin DC subset differentiation by modulating TGF-β signaling.

Cell Rep 2021 Apr;35(4):109049

Otto Loewi Research Center, Chair of Immunology and Pathophysiology, Medical University of Graz, 8010 Graz, Austria. Electronic address:

Transforming growth factor β (TGF-β) family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF-β family signaling for their differentiation, and canonical TGF-β1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). By studying mechanisms in inflammation, we previously screened LCs versus moDCs for differentially expressed microRNAs (miRNAs). This revealed that miR-424/503 is the most strongly inversely regulated (moDCs > LCs). We here demonstrate that miR-424/503 is induced during moDC differentiation and promotes moDC differentiation in human and mouse. Inversely, forced repression of miR-424 during moDC differentiation facilitates TGF-β1-dependent LC differentiation. Mechanistically, miR-424/503 deficiency in monocyte/DC precursors leads to the induction of TGF-β1 response genes critical for LC differentiation. Therefore, the miR-424/503 gene cluster plays a decisive role in anti-inflammatory LC versus pro-inflammatory moDC differentiation from monocytes.
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http://dx.doi.org/10.1016/j.celrep.2021.109049DOI Listing
April 2021

Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL).

Sci Rep 2021 Mar 4;11(1):5154. Epub 2021 Mar 4.

Department of Computational Biology, St Jude Children's Research Hospital, 262 Danny Thomas Place, MS321, Memphis, TN, 38105, USA.

USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.
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http://dx.doi.org/10.1038/s41598-021-84647-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933146PMC
March 2021

Discovery of regulatory noncoding variants in individual cancer genomes by using cis-X.

Nat Genet 2020 08 6;52(8):811-818. Epub 2020 Jul 6.

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

We developed cis-X, a computational method for discovering regulatory noncoding variants in cancer by integrating whole-genome and transcriptome sequencing data from a single cancer sample. cis-X first finds aberrantly cis-activated genes that exhibit allele-specific expression accompanied by an elevated outlier expression. It then searches for causal noncoding variants that may introduce aberrant transcription factor binding motifs or enhancer hijacking by structural variations. Analysis of 13 T-lineage acute lymphoblastic leukemias identified a recurrent intronic variant predicted to cis-activate the TAL1 oncogene, a finding validated in vivo by chromatin immunoprecipitation sequencing of a patient-derived xenograft. Candidate oncogenes include the prolactin receptor PRLR activated by a focal deletion that removes a CTCF-insulated neighborhood boundary. cis-X may be applied to pediatric and adult solid tumors that are aneuploid and heterogeneous. In contrast to existing approaches, which require large sample cohorts, cis-X enables the discovery of regulatory noncoding variants in individual cancer genomes.
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http://dx.doi.org/10.1038/s41588-020-0659-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679232PMC
August 2020

A comparison of methods accounting for batch effects in differential expression analysis of UMI count based single cell RNA sequencing.

Comput Struct Biotechnol J 2020 30;18:861-873. Epub 2020 Mar 30.

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, United States.

Accounting for batch effects, especially latent batch effects, in differential expression (DE) analysis is critical for identifying true biological effects. Single-cell RNA sequencing (scRNA-seq) is a powerful tool for quantifying cell-to-cell variation in transcript abundance and characterizing cellular dynamics. Although many scRNA-seq DE analysis methods accommodate known batch variables, their performance has not been systematically evaluated. Moreover, the challenge of accounting for latent batch variables in scRNA-seq DE analysis is largely unmet. In contrast, many methods have been developed to account for batch variables (either known or latent) in other high-dimensional data, especially bulk RNA-seq. We extensively evaluate 11 methods for batch variables in different scRNA-seq DE analysis scenarios, with a primary focus on latent batch variables. We demonstrate that for known batch variables, incorporating them as covariates into a regression model outperformed approaches using a batch-corrected matrix. For latent batches, fixed effects models have inflated FDRs, whereas aggregation-based methods and mixed effects models have significant power loss. Surrogate variable based methods generally control the FDR well while achieving good power with small group effects. However, their performance (except that of SVA) deteriorated substantially in scenarios involving large group effects and/or group label impurity. In these settings, SVA achieves relatively good performance despite an occasionally inflated FDR (up to 0.2). Finally we make the following recommendations for scRNA-seq DE analysis: 1) incorporate known batch variables instead of using batch-corrected data; and 2) employ SVA for latent batch correction. However, better methods are still needed to fully unleash the power of scRNA-seq.
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http://dx.doi.org/10.1016/j.csbj.2020.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163294PMC
March 2020

Latent cellular analysis robustly reveals subtle diversity in large-scale single-cell RNA-seq data.

Nucleic Acids Res 2019 12;47(22):e143

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for characterizing the cell-to-cell variation and cellular dynamics in populations which appear homogeneous otherwise in basic and translational biological research. However, significant challenges arise in the analysis of scRNA-seq data, including the low signal-to-noise ratio with high data sparsity, potential batch effects, scalability problems when hundreds of thousands of cells are to be analyzed among others. The inherent complexities of scRNA-seq data and dynamic nature of cellular processes lead to suboptimal performance of many currently available algorithms, even for basic tasks such as identifying biologically meaningful heterogeneous subpopulations. In this study, we developed the Latent Cellular Analysis (LCA), a machine learning-based analytical pipeline that combines cosine-similarity measurement by latent cellular states with a graph-based clustering algorithm. LCA provides heuristic solutions for population number inference, dimension reduction, feature selection, and control of technical variations without explicit gene filtering. We show that LCA is robust, accurate, and powerful by comparison with multiple state-of-the-art computational methods when applied to large-scale real and simulated scRNA-seq data. Importantly, the ability of LCA to learn from representative subsets of the data provides scalability, thereby addressing a significant challenge posed by growing sample sizes in scRNA-seq data analysis.
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http://dx.doi.org/10.1093/nar/gkz826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902034PMC
December 2019

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia.

Nat Commun 2019 06 26;10(1):2789. Epub 2019 Jun 26.

Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

[email protected] proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between [email protected] proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.
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http://dx.doi.org/10.1038/s41467-019-10637-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594946PMC
June 2019

CNS angiogenesis and barriergenesis occur simultaneously.

Dev Biol 2017 05 29;425(2):101-108. Epub 2017 Mar 29.

Department of Chemical Biology & Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, WI, USA. Electronic address:

The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously.
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http://dx.doi.org/10.1016/j.ydbio.2017.03.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682946PMC
May 2017

Active medulloblastoma enhancers reveal subgroup-specific cellular origins.

Nature 2016 Feb 27;530(7588):57-62. Epub 2016 Jan 27.

Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, Washington 98105, USA.

Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.
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http://dx.doi.org/10.1038/nature16546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5168934PMC
February 2016

Oncogenic KRAS promotes malignant brain tumors in zebrafish.

Mol Cancer 2015 Feb 3;14:18. Epub 2015 Feb 3.

Department of Chemical Biology & Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

Background: Zebrafish have been used as a vertebrate model to study human cancers such as melanoma, rhabdomyosarcoma, liver cancer, and leukemia as well as for high-throughput screening of small molecules of therapeutic value. However, they are just emerging as a model for human brain tumors, which are among the most devastating and difficult to treat. In this study, we evaluated zebrafish as a brain tumor model by overexpressing a human version of oncogenic KRAS (KRAS(G12V)).

Methods: Using zebrafish cytokeratin 5 (krt5) and glial fibrillary acidic protein (gfap) gene promoters, we activated Ras signaling in the zebrafish central nervous system (CNS) through transient and stable transgenic overexpression. Immunohistochemical analyses were performed to identify activated pathways in the resulting brain tumors. The effects of the MEK inhibitor U0126 on oncogenic KRAS were evaluated.

Results: We demonstrated that transient transgenic expression of KRAS(G12V) in putative neural stem and/or progenitor cells induced brain tumorigenesis. When expressed under the control of the krt5 gene promoter, KRAS(G12V) induced brain tumors in ventricular zones (VZ) at low frequency. The majority of other tumors were composed mostly of spindle and epithelioid cells, reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). In contrast, when expressed under the control of the gfap gene promoter, KRAS(G12V) induced brain tumors in both VZs and brain parenchyma at higher frequency. Immunohistochemical analyses indicated prominent activation of the canonical RAS-RAF-ERK pathway, variable activation of the mTOR pathway, but no activation of the PI3K-AKT pathway. In a krt5-derived stable and inducible transgenic line, expression of oncogenic KRAS resulted in skin hyperplasia, and the MEK inhibitor U0126 effectively suppressed this pro-proliferative effects. In a gfap-derived stable and inducible line, expression of oncogenic KRAS led to significantly increased mitotic index in the spinal cord.

Conclusions: Our studies demonstrate that zebrafish could be explored to study cellular origins and molecular mechanisms of brain tumorigenesis and could also be used as a platform for studying human oncogene function and for discovering oncogenic RAS inhibitors.
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http://dx.doi.org/10.1186/s12943-015-0288-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320811PMC
February 2015

Functional and genetic analysis of choroid plexus development in zebrafish.

Front Neurosci 2014 10;8:364. Epub 2014 Nov 10.

Chemical Biology and Therapeutics, St. Jude Children's Research Hospital Memphis, TN, USA ; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison Madison, WI, USA.

The choroid plexus, an epithelial-based structure localized in the brain ventricle, is the major component of the blood-cerebrospinal fluid barrier. The choroid plexus produces the cerebrospinal fluid and regulates the components of the cerebrospinal fluid. Abnormal choroid plexus function is associated with neurodegenerative diseases, tumor formation in the choroid plexus epithelium, and hydrocephaly. In this study, we used zebrafish (Danio rerio) as a model system to understand the genetic components of choroid plexus development. We generated an enhancer trap line, Et(cp:EGFP) (sj2), that expresses enhanced green fluorescent protein (EGFP) in the choroid plexus epithelium. Using immunohistochemistry and fluorescent tracers, we demonstrated that the zebrafish choroid plexus possesses brain barrier properties such as tight junctions and transporter activity. Thus, we have established zebrafish as a functionally relevant model to study choroid plexus development. Using an unbiased approach, we performed a forward genetic dissection of the choroid plexus to identify genes essential for its formation and function. Using Et(cp:EGFP) (sj2), we isolated 10 recessive mutant lines with choroid plexus abnormalities, which were grouped into five classes based on GFP intensity, epithelial localization, and overall choroid plexus morphology. We also mapped the mutation for two mutant lines to chromosomes 4 and 21, respectively. The mutants generated in this study can be used to elucidate specific genes and signaling pathways essential for choroid plexus development, function, and/or maintenance and will provide important insights into how these genetic mutations contribute to disease.
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http://dx.doi.org/10.3389/fnins.2014.00364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226144PMC
November 2014

The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma.

Nat Genet 2014 May 6;46(5):444-450. Epub 2014 Apr 6.

Department of Pediatric Cancer Genome Project, St. Jude Children's Research Hospital, Memphis, TN 38105.

Pediatric high-grade glioma (HGG) is a devastating disease with a less than 20% survival rate 2 years after diagnosis. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs), by whole-genome, whole-exome and/or transcriptome sequencing. We identified recurrent somatic mutations in ACVR1 exclusively in DIPGs (32%), in addition to previously reported frequent somatic mutations in histone H3 genes, TP53 and ATRX, in both DIPGs and NBS-HGGs. Structural variants generating fusion genes were found in 47% of DIPGs and NBS-HGGs, with recurrent fusions involving the neurotrophin receptor genes NTRK1, NTRK2 and NTRK3 in 40% of NBS-HGGs in infants. Mutations targeting receptor tyrosine kinase-RAS-PI3K signaling, histone modification or chromatin remodeling, and cell cycle regulation were found in 68%, 73% and 59% of pediatric HGGs, respectively, including in DIPGs and NBS-HGGs. This comprehensive analysis provides insights into the unique and shared pathways driving pediatric HGG within and outside the brainstem.
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http://dx.doi.org/10.1038/ng.2938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056452PMC
May 2014

Zebrafish Cacna1fa is required for cone photoreceptor function and synaptic ribbon formation.

Hum Mol Genet 2014 Jun 12;23(11):2981-94. Epub 2014 Jan 12.

Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients.
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http://dx.doi.org/10.1093/hmg/ddu009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014194PMC
June 2014

Conditional control of gene function by an invertible gene trap in zebrafish.

Proc Natl Acad Sci U S A 2012 Sep 20;109(38):15389-94. Epub 2012 Aug 20.

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.
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http://dx.doi.org/10.1073/pnas.1206131109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458342PMC
September 2012

Co-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish.

Mol Cancer 2009 Jun 25;8:40. Epub 2009 Jun 25.

Vollum Institute, Oregon Health and Science University, Portland, Oregon 97239, USA.

The zebrafish has become an important model for cancer research. Several cancer models have been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is amenable to efficient transgenesis, zebrafish have immense potential to be used for studying interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations, however, co-expressing a constitutively active human AKT1 resulted in several tumor types, including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myxoma. All tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and that the binary transgenic approach is a useful tool for studying interaction of oncogenes and oncogenic pathways in zebrafish.
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http://dx.doi.org/10.1186/1476-4598-8-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711045PMC
June 2009

Generation of fertile and diploid fish, medaka (Oryzias latipes), from nuclear transplantation of blastula and four-somite-stage embryonic cells into nonenucleated unfertilized eggs.

Cloning Stem Cells 2005 ;7(4):255-64

Laboratory of Freshwater Fish Stocks, Bioscience and Biotechnology Center, Graduate School of Science, Nagoya University, Nagoya, Japan.

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.
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http://dx.doi.org/10.1089/clo.2005.7.255DOI Listing
April 2006

Cloning zebrafish by nuclear transfer.

Methods Cell Biol 2004 ;77:403-11

Department of Molecular, Cellular, and Developmental Biology, University of California Los Angeles, Los Angeles, California 90095, USA.

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http://dx.doi.org/10.1016/s0091-679x(04)77022-3DOI Listing
February 2005

Generation of two-color transgenic zebrafish using the green and red fluorescent protein reporter genes gfp and rfp.

Mar Biotechnol (NY) 2002 Mar;4(2):146-54

Department of Biological Sciences, National University of Singapore, Singapore 119260.

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F(1) transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.
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http://dx.doi.org/10.1007/s10126-001-0085-3DOI Listing
March 2002

Protocol for nuclear transfer in zebrafish.

Cloning Stem Cells 2003 ;5(4):333-7

Department of Molecular, Cellular, and Developmental Biology, University of California, Los Angeles, California 90095-1606, USA.

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http://dx.doi.org/10.1089/153623003772032835DOI Listing
July 2004

Development and gene expression of nuclear transplants generated by transplantation of cultured cell nuclei into non-enucleated eggs in the medaka Oryzias latipes.

Dev Growth Differ 2003 Apr;45(2):167-74

Freshwater Fish Stocks, Bioscience Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.

To develop nuclear transplantation techniques for the medaka Oryzias latipes, nuclei of cultured cells from transgenic fish were transplanted into unfertilized eggs of the orange-red variety of O. latipes, without enucleation, in two experimental series. In the first experimental series, fibroblast cells cultured from the adult caudal fin were used as donors, which carried the green fluorescent protein (GFP) gene driven by the promoter of the medaka elongation factor 1alpha-A gene. Wild-type body color was another donor genetic marker used in this experimental series. In the second experimental series, cells cultured from 6-day-old embryos were used as donors, which carried the GFP genetic marker driven by the promoter of the medaka beta-actin gene. From more than 1000 eggs transplanted in each experiment, a considerable number of nuclear transplants developed to various embryonic stages showing stage- and tissue-specific expression of the donor genetic markers, although the expression was mosaic in many cases. Three and six of the transplanted eggs in the first and second experimental series (0.3 and 0.5%, respectively) hatched, and the hatchlings expressing the genetic markers survived for up to 3 weeks. The chromosome number varied among cells in a single transplant embryo. The results obtained in these experiments may help future cloning efforts in fish.
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http://dx.doi.org/10.1034/j.1600-0854.2004.00687.xDOI Listing
April 2003

Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter.

Dev Dyn 2003 May;227(1):14-26

Department of Biological Sciences, National University of Singapore, Singapore.

A 1,934-bp muscle-specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5' promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis-elements in the promoter and that a minimal -77-bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2-gfp constructs. GFP expression in the 1,934-bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871-bp promoter. Our observation indicates that there is no distinct cis-element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP-dependent protein kinase A was injected into mylz2-gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP-expressing fast muscle. The mylz2-gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered.
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http://dx.doi.org/10.1002/dvdy.10273DOI Listing
May 2003

Cloned zebrafish by nuclear transfer from long-term-cultured cells.

Nat Biotechnol 2002 Aug 22;20(8):795-9. Epub 2002 Jul 22.

Department of Molecular, Cellular, and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095-1606, USA.

Although mammals have been cloned from genetically manipulated cultured cells, a comparable achievement has not been realized in lower vertebrates. Here we report that fertile transgenic zebrafish can be obtained by nuclear transfer using embryonic fibroblast cells from long-term cultures. The donor nuclei, modified by retroviral insertions expressing green fluorescent protein (GFP), were transplanted into manually enucleated eggs. Overall, a 2% success rate was achieved, resulting in 11 adult transgenic zebrafish expressing GFP. These nuclear transplants produced fertile, diploid offspring, and their F1/F2 progeny continued to express GFP in a pattern identical to that of the founder fish. This finding demonstrates that slowly dividing nuclei from cultured cells can be reprogrammed to support rapid embryonic development and sets up a foundation for targeted genetic manipulation in zebrafish.
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http://dx.doi.org/10.1038/nbt721DOI Listing
August 2002

Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.

Dev Dyn 2002 Mar;223(2):204-15

Department of Biological Sciences, National University of Singapore, Singapore.

A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior- and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2-kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8-GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.
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http://dx.doi.org/10.1002/dvdy.10051DOI Listing
March 2002
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