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Vladimir Korzh - One of the best experts on this subject based on the ideXlab platform.
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Development of Circumventricular Organs in the Mirror of Zebrafish Enhancer-Trap Transgenics.
Frontiers in neuroanatomy, 2017Co-Authors: Marta Garcia-lecea, Igor Kondrychyn, Evgeny Gasanov, Justyna Jedrychowska, Cathleen Teh, May-su You, Vladimir KorzhAbstract:The circumventricular organs (CVOs) are small structures lining the cavities of brain ventricular system. They are associated with the semitransparent regions of the blood-brain barrier (BBB). Hence it is thought that CVOs mediate biochemical signalling and cell exchange between the brain and systemic blood. Their classification is still controversial and development not fully understood largely due to an absence of tissue-specific molecular markers. In a search for molecular determi-nants of CVOs we studied the green fluorescent protein (GFP) expression pattern in several zebrafish Enhancer Trap transgenics including Gateways (ET33-E20) that has been instrumental in defining the development of choroid plexus. In Gateways the GFP is expressed in regions of the developing brain outside the choroid plexus, which remain to be characterized. The neuroanatomical and histological analysis suggested that some previously unassigned domains of GFP expression may correspond to at least six other CVOs – the organum vasculosum laminae terminalis, subfornical organ, paraventricular organ, pineal (epiphysis), area postrema and median eminence. Two other CVOs, parapineal and subcommissural organ were detected in other Enhancer-Trap transgenics. Hence Enhancer-Trap transgenic lines could be instrumental for developmental studies of CVOs in zebrafish and understanding of the molecular mechanism of disease such a hydrocephalus in human. Their future analysis may shed light on general and specific molecular mechanisms that regulate development of CVOs.
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zebrafish Enhancer Trap transgenic line database zeTrap 2 0
Zebrafish, 2011Co-Authors: Igor Kondrychyn, Cathleen Teh, Marta Garcialecea, Youxin Guan, Ann Kang, Vladimir KorzhAbstract:Abstract Our first Zebrafish Enhancer Trap lines database (ZETrap) generated a few years ago was a web-based system informing the scientific community about the developmental, genetic, and genomic aspects of transgenic zebrafish lines expressing the cytosolic version of EGFP. These transgenic lines were obtained in a primary screen using Tol2 transposon-mediated transgenesis. Following that, several hundreds transgenics were generated by a systematic “rejump” of the transposon from the two distinct genomic sites. This collection was expanded further by generation of transgenics expressing the membrane-tethered version of a novel red protein KillerRed. These KR transgenics are useful not only to complement the cytosolic GFP in compound GFP/KR transgenics for improved bioimaging. They also could be used to affect cells physiology by tissue-specific optogenetic generation of reactive oxygen species. We have compiled the genomic data and expression patterns of these novel ET transgenic lines in an updated onl...
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zebrafish cardiac Enhancer Trap lines new tools for in vivo studies of cardiovascular development and disease
Developmental Dynamics, 2010Co-Authors: Karlai Poon, Vladimir Korzh, Igor Kondrychyn, Michael Liebling, Marta GarcialeceaAbstract:Using the transposon-mediated Enhancer Trap (ET), we generated 18 cardiac Enhancer Trap (CET) transgenic zebrafish lines. They exhibit EGFP expression in defined cell types—the endocardium, myocardium, and epicardium—or in anatomical regions of the heart—the atrium, ventricle, valves, or bulbus arteriosus. Most of these expression domains are maintained into adulthood. The genomic locations of the transposon insertions were determined by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The expression pattern of EGFP in some CETs is unique and recapitulates expression of genes flanking the transposon insertion site. The CETs enabled us to capture the dynamics of the embryonic heart beating in vivo using fast scanning confocal microscopy coupled with image reconstruction, producing three-dimensional movies in time (4D) illustrating region-specific features of heart contraction. This collection of CET lines represents a toolbox of markers for in vivo studies of heart development, physiology, and drug screening. Developmental Dynamics 239:914–926, 2010. © 2010 Wiley-Liss, Inc.
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Transposons as tools for Enhancer Trap screens in vertebrates
Genome Biology, 2007Co-Authors: Vladimir KorzhAbstract:DNA transposons are efficient tools in transgenesis and have therefore become popular in the analysis of the regulatory genome in vertebrates via Enhancer Trap screens. Here, I discuss recent progress in this field of research, with a focus on the application of one of these transposons, namely the medaka fish derived Tol2 , to Enhancer Trapping in zebrafish, and how this approach compares with others that have a similar objective.
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zebrafish transgenic Enhancer Trap line database zeTrap
BMC Developmental Biology, 2006Co-Authors: Benjamin G H Choo, Vladimir Korzh, Igor Kondrichin, Sergey Parinov, Alexander Emelyanov, Weichang TohAbstract:Background The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective Enhancer Trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution.
Koichi Kawakami - One of the best experts on this subject based on the ideXlab platform.
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an mnr2b hlxb9lb Enhancer Trap line that labels spinal and abducens motor neurons in zebrafish
Developmental Dynamics, 2012Co-Authors: Kazuhide Asakawa, Shinichi Higashijima, Koichi KawakamiAbstract:Background: The developing nervous system consists of a variety of cell types. Animal models that allow the visualization of specific classes of neurons are crucial for the study of neuronal networks. Results: We performed an Enhancer Trap screening in zebrafish and generated a collection of transgenic lines that expressed GFP in a spatially and temporally restricted manner. Among the fish generated, we identified an insertion of the Enhancer Trap construct in the vicinity of the mnr2b/hlxb9lb gene encoding the mnx class of homeodomain transcription factor. The insertion gave rise to GFP expression predominantly in spinal motor neurons and abducens motor neurons. During embryogenesis, GFP expression was also detected in endodermal and mesodermal tissues, where mnr2b is known to be expressed. Conclusion: These results show that the Enhancer Trap construct recapitulated the expression pattern of the mnr2b gene and this transgenic line should be useful for the visualization of the spinal and abducens motor neurons in the developing nervous system. Developmental Dynamics 241:327–332, 2012. © 2011 Wiley Periodicals, Inc.
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zTrap zebrafish gene Trap and Enhancer Trap database
BMC Developmental Biology, 2010Co-Authors: Koichi Kawakami, Kazuhide Asakawa, Gembu Abe, Tokuko Asada, Ryuichi Fukuda, Aki Ito, Pradeep LalAbstract:Background We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene Trapping, Enhancer Trapping and the Gal4FF-UAS system. Gene Trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and Enhancer Trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology.
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the tol2 mediated gal4 uas method for gene and Enhancer Trapping in zebrafish
Methods, 2009Co-Authors: Kazuhide Asakawa, Koichi KawakamiAbstract:The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 Enhancer Trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene Trap and Enhancer Trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene Trap and Enhancer Trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene Trap and Enhancer Trap screens in zebrafish and their application to the studies of vertebrate neural circuits.
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analysis of genes and genome by the tol2 mediated gene and Enhancer Trap methods
Methods of Molecular Biology, 2009Co-Authors: Akihiro Urasaki, Koichi KawakamiAbstract:The Tol2 transposon system can create insertions in the zebrafish genome efficiently. By using this system, the gene Trap and Enhancer Trap methods have been developed. The gene Trap and Enhancer Trap constructs contain the green fluorescent protein (GFP) reporter gene or the yeast Gal4 transcription activator gene. By creating random integrations of these constructs in the genome, transgenic fish expressing the GFP gene or the Gal4 gene in specific cells, tissues or organs are generated. These fish are valuable resources for developmental biology. Especially, the Gal4-expressing transgenic fish can be used to ectopically express any gene of interest placed downstream of the Gal4 recognition sequence, UAS, and thereby allow visualization, modification or ablation of the Gal4-expressing cells. In this chapter, we will describe how the gene Trap and Enhancer Trap screens can be performed and how the transposon insertions created by these methods can be analyzed.
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insertional mutagenesis by the tol2 transposon mediated Enhancer Trap approach generated mutations in two developmental genes tcf7 and synembryn like
Development, 2007Co-Authors: Saori Nagayoshi, Akihiro Urasaki, Kazuhide Asakawa, Eriko Hayashi, Gembu Abe, Naoki Osato, Kazuki Horikawa, Kazuho Ikeo, Hiroyuki Takeda, Koichi KawakamiAbstract:Gene Trap and Enhancer Trap methods using transposon or retrovirus have been recently described in zebrafish. However, insertional mutants using these methods have not been reported. We report here development of an Enhancer Trap method by using the Tol2 transposable element and identification and characterization of insertional mutants. We created 73 fish lines that carried single copy insertions of an Enhancer Trap construct, which contained the zebrafish hsp70 promoter and the GFP gene, in their genome and expressed GFP in specific cells, tissues and organs, indicating that the hsp70 promoter is highly capable of responding to chromosomal Enhancers. First, we analyzed genomic DNA surrounding these insertions. Fifty-one of them were mapped onto the current version of the genomic sequence and 43% (22/51) were located within transcribed regions, either exons or introns. Then, we crossed heterozygous fish carrying the same insertions and identified two insertions that caused recessive mutant phenotypes. One disrupted the tcf7 gene, which encodes a transcription factor of the Tcf/Lef family mediating Wnt signaling, and caused shorter and wavy median fin folds and pectoral fins. We knocked down Lef1, another member of the Tcf/Lef family also expressed in the fin bud, in the tcf7 mutant, and revealed functional redundancy of these factors and their essential role in establishment of the apical ectodermal ridge (AER). The other disrupted the synembryn-like gene ( synbl ), a homolog of the C. elegans synembryn gene, and caused embryonic lethality and small pigment spots. The pigment phenotype was rescued by application of forskolin, an activator of adenylyl cyclase, suggesting that the synbl gene activates the Gα S pathway leading to activation of adenylyl cyclase. We thus demonstrated that the transposon-mediated Enhancer Trap approach can indeed create insertional mutations in developmental genes. Our present study provides a basis for the development of efficient transposon-mediated insertional mutagenesis in a vertebrate.
Jim Haseloff - One of the best experts on this subject based on the ideXlab platform.
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gal4 gfp Enhancer Trap lines for analysis of stomatal guard cell development and gene expression
Journal of Experimental Botany, 2009Co-Authors: Michael J. Gardner, Jeanmaurice Assie, Jim Haseloff, Scott R Poethig, Andrew J Baker, Alex A R WebbAbstract:To facilitate the monitoring of guard cells during development and isolation, a population of 704 GAL4 GFP Enhancer Trap lines was screened and four single insert lines with guard cell GFP expression and one with developmentally-regulated guard cell GFP expression were identified. The location of the T-DNA inserts, the expression of the flanking genes, and the promoter activity of the genomic DNA upstream of the T-DNA were characterized. The results indicated that the GFP expression pattern in at least one of the lines was due to elements in the intergenic DNA immediately upstream of the T-DNA, rather than due to the activity of the promoters of genes flanking the insert, and provide evidence for the involvement of Dof elements in regulating guard cell gene expression. It is shown further that the GAL4 GFP lines can be used to track the contribution of guard cell material in vitro, and this method was used to assess the purity of guard cell samples obtained using two methods of guard cell isolation.
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gal4 gfp Enhancer Trap lines for genetic manipulation of lateral root development in arabidopsis thaliana
Journal of Experimental Botany, 2005Co-Authors: Laurent Laplaze, Andrew J Baker, Boris Parizot, Lilian Ricaud, Alexandre Martiniere, Florence Auguy, Claudine Franche, Laurent Nussaume, Didier Bogusz, Jim HaseloffAbstract:Lateral root development occurs throughout the life of the plant and is responsible for the plasticity of the root system. In Arabidopsis thaliana, lateral root founder cells originate from pericycle cells adjacent to xylem poles. In order to study the mechanisms of lateral root development, a population of Arabidopsis GAL4-GFP Enhancer Trap lines were screened and two lines were isolated with GAL4 expression in root xylempole pericycle cells (J0121), i.e. in cells competent to become lateral root founder cells, and in young lateral root primordia (J0192). These two Enhancer Trap lines are very useful tools with which to study the molecular and cellular bases of lateral root development using targeted gene expression. These lines were used for genetic ablation experiments by targeting the expression of a toxin-encoding gene. Moreover, the molecular bases of the Enhancer Trap expression pattern were characterized. These results suggest that the lateral-root-specific GAL4 expression pattern in J0192 is due to a strong Enhancer in the promoter of the LOB-domain protein gene LBD16.
Kazuhide Asakawa - One of the best experts on this subject based on the ideXlab platform.
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an mnr2b hlxb9lb Enhancer Trap line that labels spinal and abducens motor neurons in zebrafish
Developmental Dynamics, 2012Co-Authors: Kazuhide Asakawa, Shinichi Higashijima, Koichi KawakamiAbstract:Background: The developing nervous system consists of a variety of cell types. Animal models that allow the visualization of specific classes of neurons are crucial for the study of neuronal networks. Results: We performed an Enhancer Trap screening in zebrafish and generated a collection of transgenic lines that expressed GFP in a spatially and temporally restricted manner. Among the fish generated, we identified an insertion of the Enhancer Trap construct in the vicinity of the mnr2b/hlxb9lb gene encoding the mnx class of homeodomain transcription factor. The insertion gave rise to GFP expression predominantly in spinal motor neurons and abducens motor neurons. During embryogenesis, GFP expression was also detected in endodermal and mesodermal tissues, where mnr2b is known to be expressed. Conclusion: These results show that the Enhancer Trap construct recapitulated the expression pattern of the mnr2b gene and this transgenic line should be useful for the visualization of the spinal and abducens motor neurons in the developing nervous system. Developmental Dynamics 241:327–332, 2012. © 2011 Wiley Periodicals, Inc.
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zTrap zebrafish gene Trap and Enhancer Trap database
BMC Developmental Biology, 2010Co-Authors: Koichi Kawakami, Kazuhide Asakawa, Gembu Abe, Tokuko Asada, Ryuichi Fukuda, Aki Ito, Pradeep LalAbstract:Background We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene Trapping, Enhancer Trapping and the Gal4FF-UAS system. Gene Trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and Enhancer Trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology.
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the tol2 mediated gal4 uas method for gene and Enhancer Trapping in zebrafish
Methods, 2009Co-Authors: Kazuhide Asakawa, Koichi KawakamiAbstract:The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 Enhancer Trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene Trap and Enhancer Trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene Trap and Enhancer Trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene Trap and Enhancer Trap screens in zebrafish and their application to the studies of vertebrate neural circuits.
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insertional mutagenesis by the tol2 transposon mediated Enhancer Trap approach generated mutations in two developmental genes tcf7 and synembryn like
Development, 2007Co-Authors: Saori Nagayoshi, Akihiro Urasaki, Kazuhide Asakawa, Eriko Hayashi, Gembu Abe, Naoki Osato, Kazuki Horikawa, Kazuho Ikeo, Hiroyuki Takeda, Koichi KawakamiAbstract:Gene Trap and Enhancer Trap methods using transposon or retrovirus have been recently described in zebrafish. However, insertional mutants using these methods have not been reported. We report here development of an Enhancer Trap method by using the Tol2 transposable element and identification and characterization of insertional mutants. We created 73 fish lines that carried single copy insertions of an Enhancer Trap construct, which contained the zebrafish hsp70 promoter and the GFP gene, in their genome and expressed GFP in specific cells, tissues and organs, indicating that the hsp70 promoter is highly capable of responding to chromosomal Enhancers. First, we analyzed genomic DNA surrounding these insertions. Fifty-one of them were mapped onto the current version of the genomic sequence and 43% (22/51) were located within transcribed regions, either exons or introns. Then, we crossed heterozygous fish carrying the same insertions and identified two insertions that caused recessive mutant phenotypes. One disrupted the tcf7 gene, which encodes a transcription factor of the Tcf/Lef family mediating Wnt signaling, and caused shorter and wavy median fin folds and pectoral fins. We knocked down Lef1, another member of the Tcf/Lef family also expressed in the fin bud, in the tcf7 mutant, and revealed functional redundancy of these factors and their essential role in establishment of the apical ectodermal ridge (AER). The other disrupted the synembryn-like gene ( synbl ), a homolog of the C. elegans synembryn gene, and caused embryonic lethality and small pigment spots. The pigment phenotype was rescued by application of forskolin, an activator of adenylyl cyclase, suggesting that the synbl gene activates the Gα S pathway leading to activation of adenylyl cyclase. We thus demonstrated that the transposon-mediated Enhancer Trap approach can indeed create insertional mutations in developmental genes. Our present study provides a basis for the development of efficient transposon-mediated insertional mutagenesis in a vertebrate.
Dolf Weijers - One of the best experts on this subject based on the ideXlab platform.
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cell type specific promoter identification using Enhancer Trap lines
Methods of Molecular Biology, 2018Co-Authors: Tatyana Radoeva, Shunsuke Saiga, Dolf WeijersAbstract:Many developmental processes involve transitions between different cell identities as cells differentiate or undergo reprogramming. Cell identity specifications are generally associated with the activation and suppression of specific sets of genes mediated by transcription factors. Therefore, transcriptional reporters, such as promoters of cell-type-specific genes, are broadly used as cell identity markers in developmental biology. In Arabidopsis (Arabidopsis thaliana), a collection of GAL4/UAS Enhancer Trap lines is an established standard for inferring cell identity. However, only a few of these Enhancer Trap lines have been molecularly characterized, which limits their potential. Here, we describe an approach for a detailed characterization of expression and mapping of T-DNA insert location of GAL4/UAS Enhancer Trap lines. Additionally, we demonstrate how the acquired information can be further used for the generation of novel cell-type-specific promoters as well as for genotyping of Enhancer Trap lines.
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molecular characterization of arabidopsis gal4 uas Enhancer Trap lines identifies novel cell type specific promoters
Plant Physiology, 2016Co-Authors: Tatyana Radoeva, Shunsuke Saiga, Colette Ten A Hove, Dolf WeijersAbstract:Cell-type-specific gene expression is essential to distinguish between the numerous cell types of multicellular organism. Therefore, cell-type-specific gene expression is tightly regulated and for most genes RNA transcription is the central point of control. Thus, transcriptional reporters are broadly used markers for cell identity. In Arabidopsis (Arabidopsis thaliana), a recognized standard for cell identities is a collection of GAL4/UAS Enhancer Trap lines. Yet, while greatly used, very few of them have been molecularly characterized. Here, we have selected a set of 21 frequently used GAL4/UAS Enhancer Trap lines for detailed characterization of expression pattern and genomic insertion position. We studied their embryonic and postembryonic expression domains and grouped them into three groups (early embryo development, late embryo development, and embryonic root apical meristem lines) based on their dominant expression. We show that some of the analyzed lines are expressed in a domain often broader than the one that is reported. Additionally, we present an overview of the location of the T-DNA inserts of all lines, with one exception. Finally, we demonstrate how the obtained information can be used for generating novel cell-type-specific marker lines and for genotyping Enhancer Trap lines. The knowledge could therefore support the extensive use of these valuable lines.