Functional protein expression in codon-modified versions of genes for zebrafish. (a) Schematic of microinjection of mRNA into embryos for testing sequence features on protein expression. Protein is extracted at 24 hpf and analyzed by fluorescent western blot. Similarly, schematics in (d–g) indicate the genotype of the embryos and composition of the injection mix. (b–c) Cer modification, tested by microinjection of Cer or Cer.zf1 mRNA together with TagRFPT mRNA as a control into wildtype embryos (N = 3 groups each version). (b) Epifluorescent images of 24 hpf embryos expressing standard Cer or the version modified for zebrafish (Cer.zf1) and the matched TagRFPT co-injected control. (c) Quantification by western blot using an anti-EGFP antibody that recognizes an epitope also present in Cerulean, and an anti-TagRFPT antibody. Cer expression is the ratio of Cer and TagRFPT band intensities. (d) TagRFPT modification, tested by injection into wildtype embryos (N = 3). Experimental procedure was as in (a) except using mRNA encoding TagRFPT or TagRFPT.zf1, and Cerulean mRNA as control. Quantification by western blot with anti-TagRFPT and anti-EGFP. TagRFPT expression is the ratio of TagRFPT and Cer band intensities. (e) Cre modification, tested by injection into transgenic bActin:lox-GFP-stop-lox-RFP embryos (N = 3). Quantification by western blot with anti-TagRFPT, normalized to anti-α-tubulin. Increased RFP expression indicates greater Cre recombinase activity. (f) Gal4ff modification, tested by injection into transgenic UAS:GFP embryos together with TagRFPT mRNA as a control (N = 6). Quantification by western blot with anti-EGFP and anti-TagRFPT. GFP expression is the ratio of GFP and TagRFPT band intensities. (g) Nfsb modification, tested by injection into wildtype embryos treated with 10 mM metronidazole (met.) overnight. The fraction of embryos either dead or severely deformed is indicated (gray bars). Total number of embryos examined is indicated in italics. (h) Tol1 modification, tested by injection of Tol1 mRNA together with a plasmid containing a cassette with the β-actin promoter driving GFP, flanked by Tol1 transposon arms (N = 8). Middle: Epifluorescent images of GFP in 5 dpf embryos generated using standard Tol1 (top panel) and the zebrafish codon-modified versions (Tol1.zf1; bottom panel). Right: Quantification by western blot with anti-GFP, normalized to anti-α-tubulin. *P < 0.05.