Generation of Tg(<i>MYCN</i>:HSE:EGFP) zebrafish line.

<p>(A) Schematic diagram of the structure of PSGH2/MYCN recombinant plasmid. A mouse-MYCN fragment was cloned from HA-MYCN plasmid and inserted into the EcoRI and EcoRV sites of the PSGH2 vector. (B) A schematic presentation of the heat shock element (HSE) promoter. The artificial promoter contains eight multimerized heat shock elements flanked by two minimal promoters in opposed orientation (black arrowhead). EGFP and <i>MYCN</i> are expressed from the bidirectional promoter. The vector is flanked by I-SceI meganuclease sites (arrows). pA, SV40 polyadenylation signal. (C) Transgenic verification by qRT-PCR: M: TAKARA DL2000 marker; lane 1: Blank control (double distilled water); lane 2 and 3: WT and Tg F1 generation embryos at 3 dpf, respectively; lane 4: Positive control (plasmid). (D) Transgenic verification by westernblot: lane 1: WT embryo at 3 dpf; lane 2 and 3: Tg F1 and F2 generation embryos at 3 dpf, respectively. (E–F) EGFP (+) F0 mosaic zebrafish at 24 hours (×50) and 60 days post microinjection (×7.5). (G–H) EGFP (+) F1 Tg zebrafish at 24 hpf (×50) and 60 dpf (×10). (I) Expression of total <i>MYCN</i> (murine exogenous and zebrafish endogenous expression), which was increased gradually in Tg F1, F2 generation fish comparing with that in WT. (J) Expression of <i>NDRG1</i>, which is negative controlled by <i>MYCN</i> in human, was keeping a low lever in Tg F1 and F2 generation. **, P<0.01