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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An early requirement for nkx2.5 ensures the first and second heart field ventricular identity and cardiac function into adulthood.
TL;DR: The results support a model in which nkx genes induce downstream targets that are necessary to maintain chamber-specific identity in both early- and late-differentiating cardiomyocytes at discrete stages in cardiac morphogenesis, and shed new light on the stage-dependent mechanisms that sculpt chamber- specificCardiomyocyte identities.
Journal ArticleDOI
RAF1 mutations in childhood-onset dilated cardiomyopathy
Perundurai S. Dhandapany,Perundurai S. Dhandapany,Abdur Razzaque,Uthiralingam Muthusami,Sreejith Kunnoth,Jonathan J. Edwards,Sonia Mulero-Navarro,Ilan Riess,Sherly Pardo,Jipo Sheng,Deepa Selvi Rani,Bindhu Rani,Periyasamy Govindaraj,Elisabetta Flex,Tomohiro Yokota,Michiko Furutani,Tsutomu Nishizawa,Toshio Nakanishi,Jeffrey Robbins,Giuseppe Limongelli,Roger J. Hajjar,Djamel Lebeche,Ajay Bahl,Madhu Khullar,Andiappan Rathinavel,Kirsten C. Sadler,Marco Tartaglia,Rumiko Matsuoka,Kumarasamy Thangaraj,Bruce D. Gelb,Bruce D. Gelb +30 more
TL;DR: Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner, which resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin.
Journal ArticleDOI
Identification and characterization of T reg-like cells in zebrafish.
Melissa Kasheta,Corrie A. Painter,Finola E. Moore,Riadh Lobbardi,Alysia Bryll,Eli Freiman,David L. Stachura,Arlin B. Rogers,Yariv Houvras,David M. Langenau,Craig J. Ceol +10 more
TL;DR: This study identifies T reg–like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.
Journal ArticleDOI
tmem33 is essential for VEGF-mediated endothelial calcium oscillations and angiogenesis.
Aaron M Savage,Sathishkumar Kurusamy,Yan Chen,Zhen Jiang,Karishma Chhabria,Ryan B. MacDonald,Hyejeong R. Kim,Heather L. Wilson,Fredericus J.M. van Eeden,Angel L. Armesilla,Timothy J. A. Chico,Robert N. Wilkinson +11 more
TL;DR: It is shown that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs, and a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development is revealed.
Journal ArticleDOI
Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis.
TL;DR: Data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development, indicating a previously unrecognized complexity to CMAC assembly in vivo and elucidate a novel role for NAD+ during morphogenesis.
References
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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.