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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Regulation of developing myelin sheath elongation by oligodendrocyte calcium transients in vivo.
Anna M. Krasnow,Marc C. Ford,Leonardo E Valdivia,Leonardo E Valdivia,Stephen W. Wilson,David Attwell +5 more
TL;DR: It is shown that neuronal activity raises [Ca2+]i in developing oligodendrocytes in vivo and that the rate of intracellular calcium transients regulates elongation of developing myelin sheaths.
Journal ArticleDOI
Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs
Linlin Yin,Lisette A. Maddison,Mingyu Li,Nergis Kara,Matthew C. LaFave,Gaurav K. Varshney,Shawn M. Burgess,James G. Patton,Wenbiao Chen +8 more
TL;DR: A system for one-generation multiplex conditional mutagenesis in zebrafish using transgenic expression of both cas9 and multiple single guide RNAs and sgRNAs is demonstrated.
Journal ArticleDOI
Hooking the big one: the potential of zebrafish xenotransplantation to reform cancer drug screening in the genomic era.
TL;DR: The use of zebrafish embryos in drug screening and the potential for xenotransplantation approaches to be adopted as a preclinical tool to identify and prioritize therapies for further clinical evaluation are highlighted.
Journal ArticleDOI
Hypoxia Inducible Factor Signaling Modulates Susceptibility to Mycobacterial Infection via a Nitric Oxide Dependent Mechanism
Philip M. Elks,Sabrina Brizee,Michiel van der Vaart,Sarah R. Walmsley,Fredericus J.M. van Eeden,Stephen A. Renshaw,Annemarie H. Meijer +6 more
TL;DR: Results indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing H if-2α, aids the host during early stages of Mm infection, and represents a potential target for therapeutic intervention against tuberculosis.
Journal ArticleDOI
Ca 2+ activity signatures of myelin sheath formation and growth in vivo.
TL;DR: The authors live-imaging zebrafish myelin sheath Ca2+ activity in vivo and find that high-amplitude long-duration Ca2- transients precede calpain-dependent sheath retractions while frequent low-AMplitude short-duration transients drive sheath growth.
References
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Sean Munro,Hugh R.B. Pelham +1 more
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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.