The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.
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.
Kwan
Kristen M
KM
Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, Utah, USA.
Fujimoto
Esther
E
Grabher
Clemens
C
Mangum
Benjamin D
BD
Hardy
Melissa E
ME
Campbell
Douglas S
DS
Parant
John M
JM
Yost
H Joseph
HJ
Kanki
John P
JP
Chien
Chi-Bin
CB
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004274:DNA, Recombinant / Q000235:genetics*; D018014:Gene Transfer Techniques; D005821:Genetic Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D019895:Transposases / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019895:Transposases