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
pTransgenesis: a cross-species, modular transgenesis resource
Nick R. Love,Raphael Thuret,Yaoyao Chen,Shoko Ishibashi,Nitin Sabherwal,Roberto Paredes,Juliana Alves-Silva,Karel Dorey,Anna Noble,Matthew Guille,Yoshiki Sasai,Nancy Papalopulu,Enrique Amaya +12 more
TL;DR: The pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.
Journal ArticleDOI
Transposons As Tools for Functional Genomics in Vertebrate Models.
TL;DR: A battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain- of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice, and rats are described.
Journal ArticleDOI
Targeted transgene integration overcomes variability of position effects in zebrafish
Jennifer Anne Roberts,Irene Miguel-Escalada,Katherine Joan Slovik,Kathleen Theodora Walsh,Yavor Hadzhiev,Remo Sanges,Elia Stupka,Elizabeth K. Marsh,Jorune Balciuniene,Darius Balciunas,Ferenc Müller +10 more
TL;DR: The results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgenes activity in zebrafish.
Journal ArticleDOI
Dynamics of in vivo ASC speck formation.
Paola Kuri,Nicole L. Schieber,Thomas Thumberger,Joachim Wittbrodt,Yannick Schwab,Maria Leptin,Maria Leptin +6 more
TL;DR: A live ASC reporter is generated through CRISPR/Cas9 tagging of the endogenous gene in zebrafish and sees strong ASC expression in the skin and other epithelia that act as barriers to insult.
Journal ArticleDOI
An optogenetic toolbox for unbiased discovery of functionally connected cells in neural circuits
TL;DR: A new genetic toolbox, ‘Optobow’, is presented, which enables simultaneous optogenetic activation of single neurons in zebrafish and measuring the activity of downstream neurons in the network, which should be useful for identification and manipulation of networks of interconnected neurons, even in dense neural tissues.
References
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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.