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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Journal ArticleDOI
Identification of astroglia-like cardiac nexus glia that are critical regulators of cardiac development and function.
Nina L. Kikel-Coury,Jacob P. Brandt,Isabel A. Correia,Michael R. O’Dea,Dana F. DeSantis,Felicity Sterling,Kevin T. Vaughan,Gulberk Ozcebe,Pinar Zorlutuna,Cody J. Smith +9 more
TL;DR: Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, this article identified a neural crest-derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm.
Journal ArticleDOI
A GAL4-inducible transgenic tool kit for the in vivo modulation of Rho GTPase activity in zebrafish
TL;DR: 10 transgenic lines were generated and validated, creating a versatile tool kit for the temporal‐spatial modulation of Cdc42, RhoA, and Rac1 activity in vivo, which will enable systematic analysis of Rho GTPase function in any tissue of interest.
Journal ArticleDOI
Csy4-based vector system enables conditional chimeric gene editing in zebrafish without interrupting embryogenesis.
Journal ArticleDOI
Long-lived zebrafish Rohon-Beard cells.
TL;DR: This work identifies Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follows them for 13-16 days post fertilization, finding that about 40% survive to late larval stages and displays an unusual repertoire of cell death properties.
Posted ContentDOI
Glypican4 mediates Wnt transport between germ layers via signaling filopodia
Bo Hu,Anurag Kakkerla Balaraju,Juan J Rodriguez,Yuanyuan Gao,Nhan T Nguyen,Heston Steen,Saeb Suhaib,Songhai Chen,Fang Lin +8 more
TL;DR: Endodermal signaling filopodia that expressed GFP-Gpc4 transported Wnt5b, and likely Wnt11f2, to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency and reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.
References
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Journal ArticleDOI
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Journal ArticleDOI
GAL4-VP16 is an unusually potent transcriptional activator
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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.