Schematic procedure for the generation of the transgenic zebrafish model for pancreatic beta cell regeneration. The recombinant Tol2 plasmid and transposase mRNA were co-injected into the one-cell stage of zebrafish zygotes. The injected embryos were evaluated at 2 dpf using fluorescence stereo-microscope, and EGFP positive embryos were selected. EGFP positive embryos raised as F0 generation of Tg(ins:egfp-nsfB) zebrafish. Then each one of the F0 generation adult zebrafish was crossed with a wild-type (TU stain) zebrafish to produce F1 generation.

Transposon DNA construct and Tol2 transposase mRNA for generation of Tg(ins:egfp-ntr) zebrafish. A: Plasmid map of Tol2 recombinant plasmid. B: In vitro transcription of transposase mRNA after DNase treatment (DNA size marker is Fermentas 1 kb DNA ladder).

MTZ treatment specifically depletes beta cells of and beta cell regeneration. A: Schematic diagram for cell-labeling and assessment of beta cell regeneration. B: Before MTZ treatment (3dpf). C: After 24 hrs MTZ treatment (4dpf) ablate the beta cells. D: Expression of EGFP appeared again after 2-day MTZ withdrawal (6dpf). Arrows show the beta cells. E: Quantitative analysis GFP intensities at 3 dpf, 4 dpf, and 6 dpf (n=5). ***P<0.001.

Confirmation of beta cell depletion and regeneration with quantitative gene expression. A: ins relative expression at 3 dpf, 4 dpf, and 6 dpf. B: gcga relative expression at 3 dpf, 4 dpf, and 6 dpf. mRNA levels were quantified normalized relative to eef1a1l1 mRNA expression. ***P<0.001.