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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Book ChapterDOI
Use of phage φC31 integrase as a tool for zebrafish genome manipulation.
TL;DR: Strategies using the PhiC31 integrase to mediate recombination of transgenes containing attP and attB sites in cis to excise elements with spatial and temporal specificity are described.
Journal ArticleDOI
The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo
TL;DR: The epitope-mediated MMP activation (EMMA) assay is presented and used to observe the activation of Mmp2 (gelatinase A) by endogenous mechanisms in the intact zebrafish embryo and a transgenic line of zebra fish expressing EMMAedMmp2 under control of an inducible promoter is established to facilitate further investigation into the regulation of this ubiquitous ECM-remodeling protease in vivo.
Journal ArticleDOI
Apical PtdIns(4,5)P2 is required for ciliogenesis and suppression of polycystic kidney disease
TL;DR: Xu et al. as discussed by the authors showed that apical PtdIns(4,5)P2 is essential for ciliogenesis and the prevention of polycystic kidney disease.
Journal ArticleDOI
In vitro and in vivo single myosin step-sizes in striated muscle.
TL;DR: The high-throughput quantum dot labeled actin in vitro motility assay (Qdot assay) measures motor step-size in the context of an ensemble of actomyosin interactions and the zebrafish embryo assay (Z assay) performs single myosin step- size and step-frequency assaying in vivo combining single myOSin mechanical and whole muscle physiological characterizations in one model organism.
References
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Journal ArticleDOI
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Journal ArticleDOI
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Journal ArticleDOI
GAL4-VP16 is an unusually potent transcriptional activator
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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.