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
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Cellular Requirements for Building a Retinal Neuropil
Owen Randlett,Ryan B. MacDonald,Takeshi Yoshimatsu,Alexandra D. Almeida,Sachihiro C. Suzuki,Rachel O.L. Wong,William A. Harris +6 more
TL;DR: Neither presynaptic nor postsynaptic processes are individually essential for the formation of this synaptic neuropil, and it is concluded that an IPL can form in the complete absence of BCs.
Journal ArticleDOI
Mitochondria Maintain Distinct Ca 2+ Pools in Cone Photoreceptors
Michelle M. Giarmarco,Whitney M. Cleghorn,Stephanie R. Sloat,James B. Hurley,Susan E. Brockerhoff +4 more
TL;DR: The findings indicate that mitochondria can modulate subcellular functional specialization in photoreceptors and show that mitochondrial Ca2+ uptake in photoresceptors is complex and plays an essential role in normal function.
Journal ArticleDOI
Proliferation-independent regulation of organ size by Fgf/Notch signaling
Agnė Kozlovskaja-Gumbrienė,Ren Yi,Richard Alexander,Andy Aman,Ryan Jiskra,Danielle Nagelberg,Holger Knaut,Melainia McClain,Tatjana Piotrowski +8 more
TL;DR: This work demonstrates that Notch signaling is an integral part of the Wnt and Fgf signaling feedback loop coordinating cell migration and the self-organization of rosette-shaped sensory organs in the zebrafish lateral line system and demonstrates that in the absence of patterning cues normally provided by a Wnt/FgF signaling system, rosettes stillSelf-organize in the presence of NotCh signaling.
Journal ArticleDOI
Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish.
Robert N. Wilkinson,Marco J. Koudijs,Roger Patient,Philip W. Ingham,Stefan Schulte-Merker,Stefan Schulte-Merker,Fredericus J.M. van Eeden +6 more
TL;DR: It is established that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression and a dual function of Hh is established during induction of runx1(+) HSCs.
Journal ArticleDOI
Metabolic Regulation of Inflammasome Activity Controls Embryonic Hematopoietic Stem and Progenitor Cell Production.
Jenna M. Frame,Caroline Kubaczka,Timothy Long,Virginie Esain,Rebecca Soto,Rebecca Soto,Mariam Hachimi,Ran Jing,Arkadi Shwartz,Wolfram Goessling,George Q. Daley,Trista E. North,Trista E. North +12 more
TL;DR: It is demonstrated that NLRP3 inflammasome-mediated interleukin-1-beta (IL1β) signaling drives HSPC production in response to metabolic activity and is established as a conserved metabolic sensor that expands HSPCs production in vivo and in-vitro.
References
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Journal ArticleDOI
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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.