Generation of a stable transgenic zebrafish model for PC-specific SCA1. (A) Schematic drawing (not to scale) of expression constructs for PC-specific expression of red fluorescent reporter protein mScarlet targeted to the cytoplasmic membrane due to an N-terminal fusion with a post translational palmitoylation signal derived from the zebrafish GAP43 protein. This reporter is expressed alone, or coexpressed with non-pathological [30Q] and mutant pathological [82Q] alleles of human ataxin1. Ectopic expression in tectal neurons and retinal cells was eliminated by four copies of miRNA181a target sites (4xmiR181aT) in the 3′UTR of the transgenes [25]. All bidirectional transgene cassettes are flanked by Tol1 transposase recognition sites [26]. (B) Schematic outline of screening and breeding procedure to obtain stable transgenic zebrafish of the F2-generation, which propagate PC-specific mScarlet and Atx1 expression in a Mendelian manner. (C) Analysis of GAPmScarlet expression (a–c) of carriers crossed into the transgenic Tg(−7.5ca8:GFP)bz12 background with PC-specific cytoplasmic EGFP expression (d–f) and merged images (g–i) to confirm PC-specific coexpression of the transgene cassette in F2 larvae at 4 dpf of the established transgenic control mScarlet (upper row), Atx1[30Q] (middle row), and Atx1[82Q] (lower row) strains, respectively. The individual images display dorsal views of the larval corpus cerebelli, anterior is to the left. Scale bar: 50 µm.
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