Transgenic Zebrafish With Green Pancreatic Beta-cell And Their Application

Objective: Using transgenic technologies and green fluorescence protein(GFP) tracing, we have established a"green"pancreaticβ-cell zebrafish model, in which the green pancreaticβ-cell can be targeted for ablation by the administration of a prodrug. This model provides a excellent animal model to allow us to screen small molecular and TCM that promote the regeneration of pancreaticβ-cells.Methods: Molecular cloning and microinjection were used to analyze theβ-cell development in an established transgenic zebrafish model.Then polymerase chain reaction(PCR),whole embryo in-situ hybridization(WISH)and living fluorescence microscopy was identified that the GFP expression was initiated and expressed in the same spatiotemporal pattern as endogenous insulin transcript. Finally, we simultaneously disrupted pancreaticβ-cell with genetics method.Results: 1.We inserted INS:nfsB-GFP fusion gene in the p-IsceI vector which was microinjected into the cytoplasm of early zebrafish embryos and established the transgenic zebrafish carrying an reporter gene.2. Fluorescence screening obtained a stable heredity F1 generation of beta cell transgenic zebrafish. PCR, WISH and living fluorescence microscopy were used to identify an exogenous INS:nfsB-GFP fuse-gene which was inserted into the zebrafish genome.3.We have exploited the power of real-time visualization ofβ-cell development in living organism. So we found that cells with green fluorescence individually aligned into a single flattened layer in the midline of the notochord at 24 hour post fertilization(24 hpf). GFP expressing cells migrated and situated on the right side at 48hpf and become an right-hand organ of zebrafish at 120hpf.4. Afer embryos was treated with 10 mM Metronidazole for 48 hours, we monitored pancreaticβ-cell can be destroyed directly when nfsB protein converted the metronidazole into cytotoxic component.Conclusion:1. Through the transgenic technology we gained green pancreaticβ-cell zebrafish model .2.WISH and genomic PCR was used to identified that GFP expression can reliable traced pancreaticβ-cell development and stable inserted genome of zebrafish.3. Through chemistry genetics method pancreaticβ-cell have been destroyed successfully in living transgenic zebrafish.4. This studies successfully constructed a tractable transgenic zebrafish line which pancreatic beta cell was destroyed. In brief, theβ-cell transgenic zebrafish provided a invaluable tool which can be used to dissect the signal pathways ofβ-cell regeneration and screen small molecular compounds and Chinese Traditional Medicine that may regenerateβ-cells though a whole-animal high throughput screen.