PUBLICATION

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

Authors
Kwan, K.M., Fujimoto, E., Grabher, C., Mangum, B.D., Hardy, M.E., Campbell, D.S., Parant, J.M., Yost, H.J., Kanki, J.P., and Chien, C.B.
ID
ZDB-PUB-071023-14
Date
2007
Source
Developmental dynamics : an official publication of the American Association of Anatomists   236(11): 3088-3099 (Journal)
Registered Authors
Campbell, Douglas, Chien, Chi-Bin, Grabher, Clemens, Hardy, Melissa, Kanki, John, Kwan, Kristen, Mangum, Ben, Parant, John, Yost, H. Joseph
Keywords
transposon, EGFP, mCherry, EMCV IRES, bicistronic, nuclear localization signal
MeSH Terms
  • Animals
  • Animals, Genetically Modified*
  • Cloning, Molecular/methods*
  • DNA Transposable Elements*
  • DNA, Recombinant/genetics*
  • Gene Transfer Techniques*
  • Genetic Techniques
  • Genetic Vectors
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Plasmids/genetics
  • Recombination, Genetic
  • Transposases/metabolism
  • Zebrafish/genetics*
  • Zebrafish/metabolism
PubMed
17937395 Full text @ Dev. Dyn.
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.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping