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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Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain by scGESTALT
Bushra Raj,Daniel E. Wagner,Aaron McKenna,Shristi Pandey,Allon M. Klein,Jay Shendure,James A. Gagnon,Alexander F. Schier +7 more
TL;DR: A technology, scGESTALT, which combines cell type identification by single-cell RNA sequencing with lineage recording by cumulative barcode editing to simultaneously characterize the molecular identities and lineage histories of thousands of cells during development and disease is reported.
Journal ArticleDOI
Genetic lineage labeling in zebrafish uncovers novel neural crest contributions to the head, including gill pillar cells
Alessandro Mongera,Ajeet Pratap Singh,Mitchell P. Levesque,Yi Yen Chen,Peter Konstantinidis,Christiane Nüsslein-Volhard +5 more
TL;DR: The data point to a crucial role for the NC in enabling more efficient gas exchange, thus uncovering a novel, direct involvement of this embryonic tissue in the evolution of respiratory systems at the protochordate-vertebrate transition.
Journal ArticleDOI
Layer-specific targeting of direction-selective neurons in the zebrafish optic tectum.
TL;DR: A close correspondence between the preferred direction (PD) and the morphology of DS cells in the optic tectum is reported and suggests a simple mechanism for how tectal neurons acquire directional tuning in a nascent circuit.
Journal ArticleDOI
Cftr controls lumen expansion and function of Kupffer's vesicle in zebrafish
TL;DR: A new role for cftr is uncovered in KV morphogenesis and function during zebrafish development, and pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen, and inhibition of ion gradient formation impaired KVLumen inflation.
Genetic models of cancer in zebrafish
TL;DR: The zebrafish is emerging as an effective system for the study of the fundamental aspects of tumorigenesis, and its strengths as an experimental system for developmental biology and a complete genome sequence and an array of tools for gene manipulation make the construction of robust, physiological zebra fish cancer models increasingly possible.
References
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Journal ArticleDOI
Stages of embryonic development of the zebrafish.
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.