Example 20
2.3 nL of a solution containing 20 μg/nL plasmid DNA and 20 μg/nL tol2 mRNA was injected into the one-cell stage embryo obtained through crossing AB with Casper zebrafish. The injected F0 embryos were raised and crossed to casper zebrafish for screening. The F1 embryos for prospective Tg(hsp70I:Cerulean-P2A-CreERT2) line and Tg(fli1:mKO2) were screened for ubiquitous Cerulean expression after heat shock for 30 min at 37° C., and mKO2 expression restricted in vasculatures, respectively. Positive individual F1 adults were subsequently outcrossed to casper zebrafish, and their offspring with casper phenotype were then used for experiments when 50% transgene transmission was observed in the subsequent generation, indicating single transgene insertions.
