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Fig. 2 Transgenic ALS zebrafish. Transgenic ALS zebrafish are generated by Tol2-mediated transgenesis. (A) Constructs include a 5′ CMV promoter, human G93A-SOD1 and a 3′ EGFP C-terminal fusion protein including a SV40 late poly A tail. Microinjection of construct cDNA in the presence of Tol2 transposase RNA facilitates efficient, random integration of the transgene via flanking Tol2 sites. (B) PCR of genomic DNA extracted from 24 hpf F1 generation embryos demonstrates germline transmission of the G93A-SOD1-GFP transgene, confirming successful generation of the transgenic lines. (C) Western immunoblotting of protein lysates from 24 hpf F1 generation embryos demonstrates expression levels of the G93A-SOD1-GFP protein in three independent clutches of transgenic embryos. (D-E) Images of an F1 embryo (D; 24 hpf) and adult (E; 3 months of age) from the G21 G93A-SOD1-GFP transgenic line demonstrate stable ubiquitous expression of the fusion protein throughout the zebrafish lifespan.