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Journal ArticleDOI

The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.

TL;DR: The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large‐scale projects testing the functions of libraries of regulatory or coding sequences.
Abstract: Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.
Citations
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Journal ArticleDOI
29 Apr 2011-PLOS ONE
TL;DR: Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined.
Abstract: When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

1,249 citations

Journal ArticleDOI
25 Mar 2010-Nature
TL;DR: The data provide the first direct evidence for the source of proliferating cardiomyocytes during zebrafish heart regeneration and indicate that stem or progenitor cells are not significantly involved in this process.
Abstract: Although mammalian hearts show almost no ability to regenerate, there is a growing initiative to determine whether existing cardiomyocytes or progenitor cells can be coaxed into eliciting a regenerative response. In contrast to mammals, several non-mammalian vertebrate species are able to regenerate their hearts, including the zebrafish, which can fully regenerate its heart after amputation of up to 20% of the ventricle. To address directly the source of newly formed cardiomyocytes during zebrafish heart regeneration, we first established a genetic strategy to trace the lineage of cardiomyocytes in the adult fish, on the basis of the Cre/lox system widely used in the mouse. Here we use this system to show that regenerated heart muscle cells are derived from the proliferation of differentiated cardiomyocytes. Furthermore, we show that proliferating cardiomyocytes undergo limited dedifferentiation characterized by the disassembly of their sarcomeric structure, detachment from one another and the expression of regulators of cell-cycle progression. Specifically, we show that the gene product of polo-like kinase 1 (plk1) is an essential component of cardiomyocyte proliferation during heart regeneration. Our data provide the first direct evidence for the source of proliferating cardiomyocytes during zebrafish heart regeneration and indicate that stem or progenitor cells are not significantly involved in this process.

1,195 citations

Journal ArticleDOI
14 Feb 2018-Nature
TL;DR: The transcriptional basis of the gradual phenotypic change along the arteriovenous axis is uncovered and unexpected cell type differences are revealed: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells.
Abstract: Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited Here, using vascular sin

1,151 citations

Journal ArticleDOI
27 Jan 2011-Blood
TL;DR: These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology and help clarify the dynamic behaviors of neutrophils and macrophages after wounding.

837 citations

References
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Journal ArticleDOI
TL;DR: A series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio is described, providing for flexibility and continued evolution of the staging series as the authors learn more about development in this species.
Abstract: We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.

10,612 citations


"The Tol2kit: a multisite gateway-ba..." refers methods in this paper

  • ...Embryos were raised at 28.5°C and staged according to time postfertilization and morphology ( Kimmel et al., 1995 )....

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  • ...Embryos were raised at 28.5°C and staged according to time postfertilization and morphology (Kimmel et al., 1995)....

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Journal ArticleDOI
TL;DR: The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1, and three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
Abstract: Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

4,607 citations


"The Tol2kit: a multisite gateway-ba..." refers background in this paper

  • ...The two fluorescent proteins used were EGFP (Zhang et al., 1996) and the monomeric red fluorescent protein mCherry ( Shaner et al., 2004; Gray et al., 2006)....

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  • ...…have not been able to visualize the mCherry protein, likely for three reasons: the relative dimness of mCherry compared with EGFP, the low expression level driven by the IRES, and suboptimal imaging using 543-nm excitation, far from the peak of mCherry excitation at 587 nm (Shaner et al., 2004)....

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  • ...We have also generated a set of equivalent IRES constructs driving mCherry, but have not been able to visualize the mCherry protein, likely for three reasons: the relative dimness of mCherry compared with EGFP, the low expression level driven by the IRES, and suboptimal imaging using 543-nm excitation, far from the peak of mCherry excitation at 587 nm ( Shaner et al., 2004 )....

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  • ...The two fluorescent proteins used were EGFP (Zhang et al., 1996) and the monomeric red fluorescent protein mCherry (Shaner et al., 2004; Gray et al., 2006)....

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Journal ArticleDOI
18 Jul 1986-Cell
TL;DR: A cDNA clone is characterized that encodes a protein related to the 70 kd heat shock protein, but is expressed in normal rat liver, and it is identical with two previously described proteins: GRP78, whose synthesis is induced by glucose starvation, and BiP, which is found bound to immunoglobulin heavy chains in pre-B cells.

1,401 citations


"The Tol2kit: a multisite gateway-ba..." refers background in this paper

  • ...Three of the 3 entry clones encode such tags in the standard Gateway reading frame: p3E-MTpA encodes a 6 myc epitope tag ( Munro and Pelham, 1986; Roth et al., 1991) derived from pCS2MT; p3E-EGF-PpA encodes an EGFP tag; and p3EmCherrypA encodes an mCherry tag....

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Journal ArticleDOI
06 Oct 1988-Nature
TL;DR: It is shown that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene, and suggested that the activating region of VP16 may be near-maximally potent.
Abstract: Recent work has defined a class of transcriptional activators1–5, members of which activate transcription in yeast, plant, insect and mammalian cells6–9. These proteins contain two parts: one directs DNA binding and the other, called the activating region, presumably interacts with some component of the transcriptional machinery. Activating regions are typically acidic and require some poorly-understood aspect of structure, probably at least in part an α-helix1–5,10. Here we describe a new member of this class, formed by fusing a DNA-binding fragment of the yeast activator GAL4 to a highly acidic portion of the herpes simplex virus protein VP16 (ref. 11; also called Vmw65). VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters11–15. We show that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene. We suggest that the activating region of VP16 may be near-maximally potent and that it is not coincidental that such a strong activator is encoded by a virus.

1,312 citations


"The Tol2kit: a multisite gateway-ba..." refers background in this paper

  • ...The final middle entry clone encodes Gal4VP16, a fusion of the Gal4 DNA binding domain to the highly acidic region of the herpes simplex virus protein VP16, which strongly activates transcription downstream of the Gal4 UAS ( Sadowski et al., 1988 )....

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  • ...The Fse-Asc cassette was generated by annealing DNA oligos, and the UAS insert was conventionally cloned from pBUAS-E1b-RFP (gift of R. Köster)....

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  • ...The final middle entry clone encodes Gal4VP16, a fusion of the Gal4 DNA binding domain to the highly acidic region of the herpes simplex virus protein VP16, which strongly activates transcription downstream of the Gal4 UAS (Sadowski et al., 1988)....

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  • ...The p5E-UAS contains a 10 UAS multimerized Gal4 upstream activating sequence element from yeast, followed by an adenovirus E1b TATA box and a carp beta-actin 5 -UTR fragment (Rorth, 1996; Köster and Fraser, 2001)....

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  • ...This cassette is designed to be a transgenesis marker, particularly useful when the transgene of interest is nonfluorescent, difficult to visualize (e.g., a few neurons in the brain), or conditionally expressed (e.g., driven by a UAS sequence or a heatshock promoter)....

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Journal ArticleDOI
TL;DR: A method called recombinational cloning is described that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency.
Abstract: As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

1,085 citations


"The Tol2kit: a multisite gateway-ba..." refers methods in this paper

  • ...First, recombination-based cloning using the multisite Gateway system (Hartley et al., 2000; Cheo et al., 2004) greatly simplifies the generation of expression constructs....

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  • ...Gateway cloning technology is based on the att site-specific recombination system from lambda phage (Hartley et al., 2000)....

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