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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Book ChapterDOI
Transgenic zebrafish using transposable elements.
TL;DR: This work describes methods with a high success rate of generating transgenic zebrafish using Tol2 vectors, including key quality control steps during the transgenesis process, and presents common transposon and transposase vectors within the field based on the Tol2 system.
Journal ArticleDOI
Lysosome-Rich Enterocytes Mediate Protein Absorption in the Vertebrate Gut
Jieun Park,Daniel S. Levic,Kaelyn D. Sumigray,Jennifer Bagwell,Oznur Eroglu,Carina L. Block,Cagla Eroglu,Robert J. Barry,Colin R. Lickwar,John F. Rawls,Stephen A. Watts,Terry Lechler,Michel Bagnat +12 more
TL;DR: It is shown that specialized intestinal cells, which are called lysosome-rich enterocytes (LREs), internalize dietary protein via receptor-mediated and fluid-phase endocytosis for intracellular digestion and trans-cellular transport, which is crucial for vertebrate growth and survival.
Journal ArticleDOI
Filamin C is a highly dynamic protein associated with fast repair of myofibrillar microdamage
Yvonne Leber,Avnika A. Ruparelia,Gregor Kirfel,Peter F.M. van der Ven,Bernd Hoffmann,Rudolf Merkel,Rudolf Merkel,Robert J. Bryson-Richardson,Dieter O. Fürst +8 more
TL;DR: It is demonstrated by fluorescence recovery after photobleaching that a large fraction of FLNc is highly mobile in cultured neonatal mouse cardiomyocytes and in cardiac and skeletal muscles of live transgenic zebrafish embryos and this results help to better understand the pathomechanisms and pathophysiology of early stages ofFLNc-related myofibrillar myopathy and skeletal and cardiac diseases preceding pathological protein aggregation.
Journal ArticleDOI
Homeodomain Protein Otp and Activity-Dependent Splicing Modulate Neuronal Adaptation to Stress
Liat Amir-Zilberstein,Janna Blechman,Yehezkel Sztainberg,Yehezkel Sztainberg,William H. J. Norton,Adriana Reuveny,Nataliya Borodovsky,Maayan Tahor,Joshua L. Bonkowsky,Laure Bally-Cuif,Alon Chen,Gil Levkowitz +11 more
TL;DR: This study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing.
Journal ArticleDOI
Equalization of odor representations by a network of electrically coupled inhibitory interneurons
TL;DR: It is found that odor representations in the olfactory bulb were stabilized by interneurons that were densely coupled to the output neurons by electrical and GABAergic synapses, which stabilized combinatorial sensory representations against variations in stimulus intensity by generic mechanisms.
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.
Journal ArticleDOI
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.