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Fig. 10 Generation of transgenic parkin zebrafish.
(A) A Gal4/UAS-based bidirectional expression system consisting of two constructs was used for the generation of transgenic parkin zebrafish. The driver construct contains the ubiquitous promoter EF1α, driving the expression of Gal4/VP16, which binds to the upstream activating sequence (UAS) of the responder construct. Activation of the UAS leads to the expression of both full-length parkin and DsRed. (B) DsRed fluorescence in living offspring from transgenic zebrafish outcrossed with wildtype zebrafish. One-day-old transgenic zebrafish characterized by ubiquitous expression of DsRed can be distinguished from their non-transgenic siblings. (C) Parkin and DsRed show an overlapping expression pattern in transgenic zebrafish. Cryosections of the head of an one-day-old transgenic parkin zebrafish were analyzed for parkin expression (immunostaining with the anti-parkin mAb PRK8) and DsRed fluorescence. Cell nuclei were stained by DAPI. (D) Western blot analysis of transgenic parkin zebrafish and their non-transgenic siblings (control). Embryos were lysed at different time points and parkin present in the lysates was detected by immunoblotting using the anti-parkin mAb PRK8. hpf: hours post fertilization.