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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A novel myelin protein zero transgenic zebrafish designed for rapid readout of in vivo myelination.
TL;DR: It is demonstrated that the Tg(mpz:mEGFP) reporter line responds effectively to positive and negative small molecule regulators of myelination, and could be useful for identifying candidate drugs that specifically target myelin membrane production in vivo.
Journal ArticleDOI
Asymmetric Hapln1a drives regionalised cardiac ECM expansion and promotes heart morphogenesis in zebrafish development.
Christopher J Derrick,Juliana Sánchez-Posada,Farah Hussein,Federico Tessadori,Eric J. G. Pollitt,Aaron M Savage,Robert N. Wilkinson,Timothy J. A. Chico,Fredericus J.M. van Eeden,Jeroen Bakkers,Emily S. Noël +10 more
TL;DR: In this article, the authors identify a regionalised ECM expansion in the heart tube which promotes correct heart development, and propose a novel model whereby embryonic laterality cues orient the axis of ECM asymmetry, suggesting these two pathways interact to promote robust cardiac morphogenesis.
Journal ArticleDOI
From Stripes to a Beating Heart: Early Cardiac Development in Zebrafish
TL;DR: The zebrafish model provides an accessible vertebrate system to study early heart morphogenesis and to gain new insights into the mechanisms of congenital disease by integrating the core processes and cellular lineages central to cardiac development across vertebrates.
Journal ArticleDOI
Stathmin-like 4 is critical for the maintenance of neural progenitor cells in dorsal midbrain of zebrafish larvae.
Meng-Ju Lin,Shyh-Jye Lee +1 more
TL;DR: The results suggest that the Wnt-mediated Stmn4 homeostasis is crucial for preventing dorsal midbrain from premature differentiation via the G2 phase control during the neural keel stage.
Journal ArticleDOI
Genetic approaches to retinal research in zebrafish
TL;DR: This review focuses on genetic techniques, such as transgenic approaches and the revolutionizing new possibilities in genome editing, which are creating new possibilities and methods to study zebrafish development and function.
References
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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.