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 CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish
TL;DR: A simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish and constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies inZebrafish.
Journal ArticleDOI
Directional tissue migration through a self-generated chemokine gradient
Erika Donà,Joseph Barry,Guillaume Valentin,Guillaume Valentin,Charlotte Quirin,Anton Khmelinskii,Andreas Kunze,Sevi Durdu,Lionel R. Newton,Ana Fernandez-Minan,Ana Fernandez-Minan,Wolfgang Huber,Michael Knop,Darren Gilmour +13 more
TL;DR: The in vivo dynamics of one key guidance molecule, the chemokine Cxcl12a, are defined by applying a fluorescent timer approach to measure ligand-triggered receptor turnover in living animals and this study provides the first in vivo proof for self-directed tissue migration through local shaping of an extracellular cue.
Journal ArticleDOI
A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation
Charles K. Kaufman,Christian Mosimann,Zi Peng Fan,Song Yang,Song Yang,Andrew Thomas,Julien Ablain,Julien Ablain,Justin L. Tan,Rachel D. Fogley,Ellen van Rooijen,Ellen van Rooijen,Elliott J. Hagedorn,Elliott J. Hagedorn,Christie Ciarlo,Christie Ciarlo,Richard M. White,Dominick A. Matos,Ann-Christin Puller,Cristina Santoriello,Cristina Santoriello,Eric C. Liao,Richard A. Young,Leonard I. Zon +23 more
TL;DR: A crestin:EGFP reporter is developed that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors, and shows the importance of NCP-state reemergence as a key event in melanomas initiation from a field of cancer-prone melanocytes.
Journal ArticleDOI
A model 450 million years in the making: zebrafish and vertebrate immunity.
TL;DR: Zebrafish immunity is reviewed with a particular focus on recent studies that exploit the unique genetic and in vivo imaging advantages available for this organism, which are driving forward the study of vertebrate immunity in general.
Journal ArticleDOI
Synaptic vesicle release regulates myelin sheath number of individual oligodendrocytes in vivo
Sigrid Mensch,Marion Baraban,Rafael G. Almeida,Tim Czopka,Tim Czopka,Jessica Ausborn,Abdeljabbar El Manira,David A. Lyons +7 more
TL;DR: It is found that blocking synaptic vesicle release impaired CNS myelination by reducing the number of myelin sheaths made by individual oligodendrocytes during their short period of formation, and stimulating neuronal activity increased myelinating capacity of single oligodends.
References
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Sean Munro,Hugh R.B. Pelham +1 more
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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.