| Purpose: Subject to the use of transgenic technology, the construction of livingβ-cell green fluorescent zebra fish can trace the line, thus on this basis, further underminingβ-cell, the establishment of the isletβliving cells can trace, for high-throughput screening of diabetesβ-cell damage model tomake preparations.Methods: Through transgenic technology, PCR technology, molecular cloning methods, the establishment of GFP marker islet B-cell genetics of transgenic zebrafish strains; live Ticheng like dynamic tracing the origin of pancreatic B cells and mature in situ hybridization and the whole embryo Gene Group PCR GFP confirmed positive endogenous islet cells andβcells were targeted.Results: 1.INS was part of the promoter cloning and recombinant INS: GFP, the establishment of reproductive microscope injection of insulin transgenic zebrafish.2.Through the reproductive system of transgenic zebrafish screening and fluorescence observation, 24 hpf GFP marker of pancreaticβcells were detected in the second and third in the bottom of the center line on both sides of the oval-shaped single entity. With the development of zebrafish embryos,the pancreas gradually increasing and the right move, 72 hpf observed pancreaticβ-cell lines in the womb to the right. Disappeared after the yolk, 120 hpf clear that the pancreas right side, will move to the bottom of the duodenum dorsal fish.3.Genome-wide PCR confirmed GFP in the genome of the stability of integration, all the embryos that live in situ hybridization of the transgenic zebrafish GFP islet cells forβ-cells.Conclusion:1.The subject of transgenic technology, build aβ-cells can trace of the zebra fish. 2. Whole embryo through PCR and in situ hybridization confirmed that the method established by theβ-cells can trace of the zebra fish is completely successful.3. The success of this study to further establish high-throughput screening for the destruction ofβ-cell zebrafish disease model has laid a foundation for research. |
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