Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos
Transcrição
Begin with viable, one-cell stage zebrafish embryos placed in the wells of a microinjection mold.
Load a microinjection needle with a solution containing Tol2 transposase-encoding mRNA and transposon donor plasmids. The plasmids comprise a ubiquitous zebrafish promoter and a fluorescent protein-encoding gene flanked by Tol2 transposon arms.
Carefully inject the microinjection solution into the cytoplasm of the zebrafish embryo. Within the cell, Tol2 transposase mRNAs translate into Tol2 transposase proteins and are actively transported into the nucleus.
Tol2 sites on the donor plasmid comprise specific inverted terminal repeats, or ITRs, flanking the reporter gene. The ITRs are flanked by short, direct repeats. Transposase proteins recognize and bind to the ITR sites, forming a hairpin on the flanking DNA. Transposases further cleave the transposon construct from the flanking DNA, releasing it from the plasmid and leaving behind short, direct repeats.
Transposases create a staggered double-strand break in the somatic cell genome and insert the transposon construct into the genome site. The gap filling of the staggered ends by DNA polymerase followed by ligation of the nicks create short, direct repeats.
The ligation causes stable transposon construct integration into the somatic cell genome, and the promoter facilitates fluorescent protein expression. Incubate the injected embryos. During embryonic development, somatic cells give rise to different cell types and create transgenic zebrafish.
