Fig. 2.

Description of miniCoopR-I vectors for the doxycycline-inducible overexpression of negative and positive modulators of pigmentation. (A) Cartoon describing the general functioning of miniCoopR-I vector. mitfa promoter drives the expression of the reverse tetracycline-controlled transactivator (rtTA, grey). The gene of interest (a pigmentation modulator, brown) is located downstream of tetracycline responsive element (TRE) and gets expressed only when doxycycline (dox, yellow) is added to fish water, so that it can bind to and activate the rtTA. (B) Schematic representation of the experimental protocol to be followed to study the pigmentation modulator of interest. 20–30 pg of miniCoopR-I vector are injected together with 20–30 pg of Tol2 transposase mRNA in 1-cell stage Tg(mitfa:BRAFV600E);p53;mitfa−/− embryos. In line with the protocol described in (Ceol et al., 2011; Iyengar et al., 2012; Painter and Ceol, 2014), successfully injected embryos are selected at 48 hpf on the basis of the presence of rescued melanocytes. Then, at 2 months of age, they are selected on the basis of the presence of nevi ≥4 mm². At this time point, selected fish are treated with dox (10 uM in fish water), irrespectively of sex. At 3–8 months of age, the indicated parameters are analyzed with the appropriate techniques. (C) Cartoon describing the general functioning of miniCoopR-I-sponge-204/SCR (miR-204 inhibition) and of miniCoopR-pre-miR-204/SCR (miR-204 overexpression).