Journal ArticleDOI
The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.
Kristen M. Kwan,Esther Fujimoto,Clemens Grabher,Benjamin D. Mangum,Melissa Hardy,Douglas Simon Campbell,John M. Parant,H. Joseph Yost,John P. Kanki,Chi Bin Chien +9 more
TLDR
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.read more
Citations
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Activation of hypoxia-inducible factor-1α (Hif-1α) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model
Philip M. Elks,Fredericus J.M. van Eeden,Giles Dixon,Xingang Wang,Constantino Carlos Reyes-Aldasoro,Philip W. Ingham,Moira K. B. Whyte,Sarah R. Walmsley,Stephen A. Renshaw +8 more
TL;DR: It is demonstrated that Hif-1α regulates neutrophil function in complex ways during inflammation resolution in vivo, and site-directed mutagenesis of Hif in vivo reveals that hydroxylation of HIF-1 α by prolyl hydoxylases critically regulates the Hif pathway in zebrafish neutrophils.
Journal ArticleDOI
Distinct Cellular Mechanisms of Blood Vessel Fusion in the Zebrafish Embryo
Lukas Herwig,Yannick Blum,Alice Krudewig,Elin Ellertsdottir,Anna Lenard,Heinz-Georg Belting,Markus Affolter +6 more
TL;DR: These life observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes.
Journal ArticleDOI
Tension-oriented cell divisions limit anisotropic tissue tension in epithelial spreading during zebrafish epiboly
Pedro Campinho,Martin Behrndt,Jonas Ranft,Thomas Risler,Nicolas Minc,Carl-Philipp Heisenberg +5 more
TL;DR: It is found that in the absence of tension-oriented cell divisions and in the presence of increased tissue tension, EVL cells undergo ectopic fusions, suggesting that the reduction of tension anisotropy by oriented cell divisions is required to preventEVL cells from fusing.
Journal ArticleDOI
A transitional extracellular matrix instructs cell behavior during muscle regeneration.
TL;DR: In vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion.
Journal ArticleDOI
Optical control of metabotropic glutamate receptors
Joshua Levitz,Carlos R. Pantoja,Benjamin M. Gaub,Harald Janovjak,Harald Janovjak,Andreas Reiner,Adam Hoagland,David Schoppik,Brian Kane,Philipp Stawski,Alexander F. Schier,Dirk Trauner,Dirk Trauner,Ehud Y. Isacoff +13 more
TL;DR: Light gated two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release are light gated, paving the way for determining the roles of g protein–coupled receptors in synaptic plasticity, memory and disease.
References
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Journal ArticleDOI
Stages of embryonic development of the zebrafish.
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.
Journal ArticleDOI
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
Nathan C. Shaner,Robert E. Campbell,Robert E. Campbell,Paul Steinbach,Ben N G Giepmans,Amy E. Palmer,Roger Y. Tsien +6 more
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.
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An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein.
Sean Munro,Hugh R.B. Pelham +1 more
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.
Journal ArticleDOI
GAL4-VP16 is an unusually potent transcriptional activator
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.
Journal ArticleDOI
DNA Cloning Using In Vitro Site-Specific Recombination
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.