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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The LIM-homeodomain transcription factor Islet2a promotes angioblast migration
Ryan E. Lamont,Chang-Yi Wu,Jae.-Ryeon. Ryu,Wendy Vu,Paniz Davari,Ryan E. Sobering,Regan M. Kennedy,Nicole M. Munsie,Sarah J. Childs +8 more
TL;DR: Isl2a may act as an intermediate between Notch signaling and genetic programs controlling angioblast number and migration, placing it as a novel transcriptional regulator of early angiogenesis, and is demonstrated to be required cell autonomously in angioblasts to promote their incorporation into the vein, and are permissive for vein identity.
Journal ArticleDOI
In vivo aggregation of presynaptic alpha-synuclein is not influenced by its phosphorylation at serine-129.
Leah J. Weston,Zoe T. Cook,Teresa L. Stackhouse,Mehtab K. Sal,Baergen I. Schultz,Zachary J. C. Tobias,Valerie R. Osterberg,Nicole L. Brockway,Saheli Pizano,Greta Glover,Tamily A. Weissman,Vivek K. Unni +11 more
TL;DR: In this article, the authors used an in vivo zebrafish model to express several different forms of human α-synuclein and measure its aggregation in presynaptic terminals.
Journal ArticleDOI
Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain
TL;DR: The data provide a detailed lineage analysis of the dlx1a/2a and dlX5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons.
Journal ArticleDOI
Rab5c-mediated endocytic trafficking regulates hematopoietic stem and progenitor cell development via Notch and AKT signaling
TL;DR: It is reported that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos, and the interaction between Rab5C and Appl1 in the endosome is required for the survival of HE in the ventral wall of the dorsal aorta through AKt signaling.
Journal ArticleDOI
Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.
Christoph Seiler,Nichole Gebhart,Yong Zhang,Susan A. Shinton,Yue-Sheng Li,Nicola L. Ross,Xingjun Liu,Qin Li,Alison N. Bilbee,Gaurav K. Varshney,Matthew C. LaFave,Shawn M. Burgess,Jorune Balciuniene,Darius Balciunas,Richard R. Hardy,Dietmar J. Kappes,David L. Wiest,Jennifer Rhodes +17 more
TL;DR: Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.
References
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Journal ArticleDOI
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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
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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.