Fig. S2

Improvement of the pIDM system by including the combination of a pair of insulators. (A) Diagram of the first generation pIDM vector. The abbreviations for each item in the first generation pIDM construct are the same as those in the improved pIDM construct (Figure 1A), except where it lacks the pair of insulators. (B) Frequencies of F₀ founder fish with visible fluorescent F₁ progenies. F₀ transgenic founder fish were generated with either the first generation or improved pIDM constructs. F₁ population containing visible green fluorescent embryos was considered to be positive transgenic lines. (C) The line on the left presented most transgenic lines generated with the first generation pIDM and the line on the right presented most transgenic lines generated with the improved pIDM. The picture was taken at 36 hpf. Red arrow, embryo without visible fluorescence. (D) Identification of transgene by PCR using rtTA-transgenic-fish-ID-for and rtTA-transgenic-fish-ID-rev primer pairs in F₁ transgenic embryos generated with the first generation pIDM. Genomic DNA was extracted from WT embryos and F₁ embryos either presence or absence of fluorescence. The plasmid DNA of pIDM (plasmid) was used as a positive control. (E) The inclusion of the insulator in pIDM sharply decreased the leakage of the Tet-on promoter. Upper panel: Diagram showing two types of vectors under analysis, either without or with a pair of insulators. In this construct, we used DsRed as the reporter gene and Egfp as the inducible gene. Lower panels: Western blot analysis of EGFP. The plasmid was injected into WT embryos at the one-cell stage. The injected embryos were treated with Dox at 12 hpf. Total protein was extracted at 8, 18 or 24 hpt. An EGFP antibody was used to detect EGFP protein. β-actin was used as the protein loading control.