The specific details of the methods have been left out for the sake of brevity, but can be observed by referring to the attached article. First, a GFP transgenic Zebrafish was produced by injecting cesium-purified plasmid DNA and Tc3A transposease into one or two-celled embryos of the zebrafish. Bright-field microscopy was then used to observe the results. In the next step, various strains of the zebrafish were obtained and maintained. INS-GFP embryos were then grown on agarose treated dishes in embryo media and imaged. Cyclopamine treatment consisted of removal of chorions using pronase and growth on agarose-covered plates. Paraffin sections were then stained with hematoxylin and eosin, and whole-mount procedures were performed. A probe was embedded in paraffin, sectioned, and counterstained with vector green to show the presence of exocrine pancreas in mutant embryos. Insulin-hybridized mutant embryos were also sectioned. Next, the Trizol method was used to prepare total RNA from adult pancreatic tissue. Reverse transcription was used to obtain cDNA and used as a template to amplify pancreas-specific genes with the use of specific primers based on a published sequence. Finally, overexpression of Shh mRNA was performed. The expression plasmid pT7TS-shh was used to create capped Shh mRNA by using the SP6 Message Machine, and injected into yolks of embryos at the two-to four-cell stage. .
In the previously mentioned INS-GFP transgenic zebrafish, the insulin promoter targeted expression of GFP to the insulin-expressing cells of the islet. At the 17-somite stage (18hpf), GFP expression was observed in cells of the midline gut endoderm ventral to somite 4, and at 48 hpf (refer to Fig. 1 in the article), the free swimming zebrafish is seen to have an islet, as evident in the discrete cluster of GFP-positive cells. The exposure to cyclopamine resulted in blockage of islet formation, as expected from the hh signal inhibitor.