Fig. 2

The mouse “GAB” and the chicken “5′HS4” insulators are functional in zebrafish. We have generated a new Tol2 transposon-based vector to test the insulator potential of candidate sequences. This vector contains a strong midbrain-specific enhancer (Z48) 5′ of the Cardiac Actin promoter. A: These two components drive GPF expression in the midbrain (Pale orange bracket), due to activity of the Z48 enhancer, and in the somites (pale blue open bracket), due to an endogenous activity of the Cardiac Actin promoter. B: If a strong insulator is placed between the enhancer and the promoter, the midbrain expression should be reduced. C-E: When we placed either the mouse GAB or the chicken 5′HS4 insulators in this vector, we observe that the ratio between midbrain and muscle expression is decreased (D,E) compared with that observed in controls bearing the Gateway entry sequence of 1.7 kb, which has no insulator activity (C). For each insulator, the reduction of the midbrain enhancer activity was observed in 45-65% of the injected embryos (n > 100).