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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Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.
Jamie Rae Acosta,Claire Goldsbury,Claire Winnick,Andrew P. Badrock,Stuart T. Fraser,Angela S. Laird,Thomas E. Hall,Emily K. Don,Jennifer A. Fifita,Ian P. Blair,Garth A. Nicholson,Nicholas J. Cole +11 more
TL;DR: It is demonstrated that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type.
Journal ArticleDOI
The Joubert Syndrome Protein Inpp5e Controls Ciliogenesis by Regulating Phosphoinositides at the Apical Membrane
TL;DR: It is shown that Inpp5e functions as a key regulator of cell polarity in the renal epithelia, by inhibiting PtdIns(3,4,5)P3 and subsequently stabilizing PTDIns(4, 5)P2 and recruiting Ezrin, F-actin, and basal bodies to the apical membrane, and suggest a possible novel approach for treating human ciliopathies.
Journal ArticleDOI
Hedgehog signaling via angiopoietin1 is required for developmental vascular stability.
Ryan E. Lamont,Wendy Vu,Alyson D. Carter,Fabrizio C. Serluca,Calum A. MacRae,Sarah J. Childs +5 more
TL;DR: It is shown that the loss of angiopoietin1 (angpt1) expression in ventral perivascular mesenchyme is responsible for vascular instability in igu mutants, and over-expression of angpt1 or partial down-regulation of the endogenous Angpt1 antagonist angpt2 rescues hemorrhage.
Journal ArticleDOI
Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification.
TL;DR: It is concluded that regulation of dopaminergic neuron phenotype in vertebrates is regulated by dispersed regulatory elements.
Journal ArticleDOI
Slit1b-Robo3 Signaling and N-Cadherin Regulate Apical Process Retraction in Developing Retinal Ganglion Cells
TL;DR: It is shown by knockdown experiments that the guidance molecule Slit1b and its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs) and that Slit-Robo signaling downregulates N-cadherin activity to allow apical Retraction in newly generated RGCs.
References
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Journal ArticleDOI
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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.
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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.