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
Nanog-like regulates endoderm formation through the Mxtx2-nodal pathway
Cong Xu,Cong Xu,Zi Peng Fan,Patrick Müller,Rachel D. Fogley,Anthony DiBiase,Anthony DiBiase,Eirini Trompouki,Eirini Trompouki,Juli Unternaehrer,Juli Unternaehrer,Fengzhu Xiong,Ingrid Torregroza,Todd Evans,Sean G. Megason,George Q. Daley,George Q. Daley,Alexander F. Schier,Richard A. Young,Leonard I. Zon,Leonard I. Zon +20 more
TL;DR: In this article, the role of Nanog-like morphants in the formation of the extraembryonic syncytial layer (YSL) required for endoderm induction was examined.
Journal ArticleDOI
Single continuous lumen formation in the zebrafish gut is mediated by smoothened-dependent tissue remodeling.
TL;DR: During gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts, indicating that fluid-driven lumen enlargement and resolution are two distinct processes.
Journal ArticleDOI
Wnt5b–Ryk pathway provides directional signals to regulate gastrulation movement
TL;DR: The soluble ligand Wnt5b repels cells expressing the Ryk (related to tyrosine kinase) receptor, establishing directional motility during gastrulation.
Journal ArticleDOI
WldS and PGC-1α Regulate Mitochondrial Transport and Oxidation State after Axonal Injury
TL;DR: Monitoring mitochondrial morphology, transport, and redox state before and after laser axotomy indicates that mitochondrial oxidation state is a more reliable indicator of axon vulnerability to degeneration than mitochondrial motility.
Journal ArticleDOI
CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.
Gopal Kushawah,Luis Hernandez-Huertas,Joaquin Abugattas-Nuñez del Prado,Joaquin Abugattas-Nuñez del Prado,Juan R. Martinez-Morales,Michelle Lynn DeVore,Huzaifa Hassan,Ismael Moreno-Sanchez,Laura Tomas-Gallardo,Alejandro Díaz-Moscoso,Dahiana E. Monges,Javier R. Guelfo,William C. Theune,Emry O. Brannan,Wei Wang,Timothy J. Corbin,Andrea Moran,Alejandro Sánchez Alvarado,Edward Málaga-Trillo,Carter M. Takacs,Ariel A. Bazzini,Ariel A. Bazzini,Miguel A. Moreno-Mateos +22 more
TL;DR: It is shown that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos and can be used in medaka, killifish, and mouse embryos.
References
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Journal ArticleDOI
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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.