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The amplified product was digested with NotI and cloned into the NotI site of the ET construct (Parinov et al., 2004) carrying a TAA stop codon after EGFP to terminate the translation of Kras.
For in vivo remobilization of a single-copy of Tol2 transposon-based ET construct, 50-100 pg of transposase mRNA was injected into zebrafish embryos from the donor lines at the one- or two-cell stage.
The ET krt4 EGFP SqET33-E20 line (referred to as SqET33-E20) was created after in vivo remobilization of a single-copy of Tol2 transposon-based ET construct from the donor line SqET33 [ 22].
The ET krt4 EGFP SqET33 line (referred to as SqET33) was established by coinjection of in vitro synthesized transposase mRNA and Tol2 transposon-based ET construct DNA into wild type zebrafish embryos at the one- or two-cell stage [ 13].
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