LAMBDA RED-MEDIATED BAC RECOMBINEERING ALLOWS REAL-TIME VISUALIZATION OF PANCREAS DEVELOPMENT IN LIVING ZEBRAFISH EMBRYOS.

Abstract

Background: The basic helix-loop-helix transcription factor ptf1a plays a central role in the mammalian pancreatic development. In zebrafish, its expression is observed in the developing hindbrain and pancreas in a temporally and spatially restricted pattern. To establish an in vivo system for visualizing developing pancreas, we generated a bacterial artificial chromosome (BAC) construct having green fluorescence gene under the regulation of ptf1a promoter by engineering BAC through homologous recombination. Materials and Methods: The template plasmid pKD4-GFP was constructed by cloning GFP gene into pKD4 vector. BAC CH211-142H2 (Encompassing zebrafish ptf1a locus) targeting constructs were generated by PCR. Within each primer, the first 39–41 bp was homologous to the pft1a sequence on BAC, and the next 20–21 bp was complementary to the template plasmid. Bacteria carrying both BAC and pKD46 (λ Red recombinase) were transformed with targeting constructs by electroporation. Positive colonies having undergone homologous recombination replacing ptf1a-coding sequence with GFP were selected. The purified BAC construct containing the EGFP gene under the regulation of zebrafish ptf1a promoter was injected directly into the yolk of zebrafish embryos at single-cell stage. Results: Transient GFP expression was observed in hindbrain and retina at 24 hpf and in pancreas at 32 hpf. In hindbrain, the fluorescence was gradually intensified till 48 hpf, maintained for 1–2 days, and extinguished at 96 hpf. Pancreatic fluorescence was increased till 3–4 dpf and maintained for several days. The fluorescence was confined to exocrine pancreas and was excluded from the central islet. The expression rate and intensity was dose-dependent, i.e. 25 % with 1–2 ng and 50% with 3–4 ng. GFP expression was specific and reproducible, and ectopic expression was observed in less than 10%. Those fishes with fluorescence are being raised and their F1 progeny will be screened for GFP expression. Conclusions: Developing pancreas can be visualized in living zebrafish embryos and the transgenic line, once established, will provide a useful model of labeled exocrine pancreatic cells for in vivo.