The Tol2kit: A multisite gateway‐based construction kit for Tol2 transposon transgenesis constructs

2007/10/15 by Kristen M. Kwan, E. Fujimoto, Esther Fujimoto +9 · 11 citations
Biochemistry, Genetics and Molecular Biology · #Advanced Fluorescence Microscopy Techniques #CRISPR and Genetic Engineering #Zebrafish Biomedical Research Applications

paper · doi:10.1002/dvdy.21343

openalex publication_date 2007/10/15 · openalex created_date 2016/06/24 · openalex updated_date 2026/07/31

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.

Citations

• Germ‐line transmission of a myocardium‐specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish
• Gateway compatible vectors for analysis of gene function in the zebrafish
• Stages of embryonic development of the zebrafish
• Laser-induced gene expression in specific cells of transgenic zebrafish

Cited by

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• Multiple cis‐regulatory elements collaborate to control mdka expression in telencephalic neural stem cells of adult zebrafish during constitutive and regenerative neurogenesis
• Gateway compatible vectors for analysis of gene function in the zebrafish
• In vivo aggregation of presynaptic alpha-synuclein is not influenced by its phosphorylation at serine-129
• A subset of neutrophil phagosomes is characterised by pulses of Class I PI3K activity
• Quantification of subcellular RNA localization through direct detection of RNA oxidation
• MARCKS phosphorylation by PKC strongly impairs cell polarity in the chick neural plate
• Multiple cis-regulatory elements collaborate to control mdka expression in telencephalic neural stem of adult zebrafish during constitutive and regenerative neurogenesis
• A dynein-driven nucleokinesis program enables neural crest migration through confined tissues in vivo
• The sclerotome is the source of the dorsal and anal fin skeleton and its expansion is required for median fin development

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