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
Cardiac Myocyte-Specific AHR Activation Phenocopies TCDD-Induced Toxicity in Zebrafish
TL;DR: This work identifies a single cellular site of TCDD action, the myocardial cell, that can account for the severe cardiovascular collapse observed following early life stage exposure to T CDD, and contributes to other forms of toxicity.
Book ChapterDOI
Chapter 9 – Analysis of cilia structure and function in zebrafish
TL;DR: A set of protocols to examine cilia morphology, motility, and cilia-related defects in a variety of organs, focusing on the embryo and early postembryonic development is provided.
Journal ArticleDOI
Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos
TL;DR: This work shows that a loss-of-function mutation in lmo2, a gene specifically required for hematopoiesis and vascular development, results in failure of optic fissure closure in zebrafish, and reveals a new mechanism leading to coloboma, whereby malformed blood vessels interfere with eye morphogenesis.
Journal ArticleDOI
A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States.
Shauna Katz,Delphine Cussigh,Noelia Urbán,Isabelle Maria Blomfield,François Guillemot,Laure Bally-Cuif,Laure Bally-Cuif,Marion Coolen,Marion Coolen +8 more
TL;DR: It is demonstrated that in the adult zebrafish pallium, the brain-enriched miR-9 is expressed exclusively in a subset of quiescent NSCs, highlighting a heterogeneity within these cells, and proposed a conserved non-canonical role for nuclear miR/Agos in controlling the balance between NSC quiescence and activation, a key step in maintaining adult germinal pools.
Journal ArticleDOI
PCP Signaling between Migrating Neurons and their Planar-Polarized Neuroepithelial Environment Controls Filopodial Dynamics and Directional Migration
TL;DR: It is shown that PCP signaling is required both within FBMNs and the hindbrain rhombomere 4 environment at the time when they initiate their migration, and a model in whichPCP signaling between the planar polarized neuroepithelial environment and FBMNS directs migration by the selective stabilization of FBMN filopodia is suggested.
References
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Journal ArticleDOI
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Journal ArticleDOI
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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.