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
More filters
Journal ArticleDOI
Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish.
Leonard Burg,Nicholas J Palmer,Khrievono Kikhi,Evgeniya S. Miroshnik,Helen Rueckert,Eleanor Gaddy,Carlee MacPherson Cunningham,Kenny Mattonet,Shih-Lei Lai,Rubén Marín-Juez,Richard B. Waring,Didier Y.R. Stainier,Darius Balciunas +12 more
TL;DR: A simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair is developed.
Journal ArticleDOI
The N17 domain mitigates nuclear toxicity in a novel zebrafish Huntington’s disease model
Matthew B. Veldman,Yesenia Rios-Galdamez,Xiao-Hong Lu,Xiao-Hong Lu,Xiaofeng Gu,Xiaofeng Gu,Wei Qin,Song Li,X. William Yang,X. William Yang,Shuo Lin,Shuo Lin +11 more
TL;DR: Test the function of the N-terminal 17 amino acids (N17) in the context of the exon 1 fragment of HTT in a novel, inducible zebrafish model of HD to study mechanisms of HD at the behavioral, cellular, and molecular levels.
Journal ArticleDOI
Renin expression in developing zebrafish is associated with angiogenesis and requires the Notch pathway and endothelium
TL;DR: It is shown that Notch signaling and the endothelium are essential for developmental renin expression, and after inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts.
Journal ArticleDOI
Visualization of Lipid Metabolism in the Zebrafish Intestine Reveals a Relationship between NPC1L1-Mediated Cholesterol Uptake and Dietary Fatty Acid
TL;DR: It is found that a lipid-rich meal promotes BODIPY-cholesterol absorption into an endosomal compartment distinguishable from lipid droplets, and it is shown that dietary fatty acids promote intestinal cholesterol absorption by rapid re-localization of NPC1L1 to the intestinal brush border.
Journal ArticleDOI
Real-time Visualization of Oxidative Stress-Mediated Neurodegeneration of Individual Spinal Motor Neurons in Vivo
Isabel Formella,Adam J. Svahn,Rowan A. W. Radford,Emily K. Don,Nicholas J. Cole,Alison L. Hogan,Albert Lee,Roger S. Chung,Marco Morsch +8 more
TL;DR: The data demonstrate the successful implementation of KR mediated ROS toxicity in motor neurons in vivo and has important implications for studying the effects of ROS in a variety of conditions within the central nervous system, including aging and age-related neurodegenerative diseases.
References
More filters
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.