Generation of transgenic zebrafish responding to hypoxia using the To12 transposon system
Objective To generate transgenic zebrafish that can respond to hypoxia for drug screening with HIF-la as the target of action.Methods Two transposable plasmids,pT2(HRE-sv40mp-GFP)and pT2(HREM-sv40mp-GFP),were generated,and the plasmids were transfected in cells and the intracellular green fluorescent protein(GFP)expression was observed under normoxic and hypoxic conditions;the two transposable plasmids were microinjected into zebrafish embryos,hypoxia treatment of embryos and observation of green fluorescence intensity of embryos to verify the hypoxia responsiveness of transgenic zebrafish.Tg(HRE-sv40mp:GFP)and Tg(HREM-sv40mp:GFP)transgenic zebrafish were obtained after multi-generation screening;the hypoxic Tg(HRE-sv40mp:GFP)zebrafish embryos were treated with the HIF-la inhibitor LW6 or Chrysin,respectively.The embryos were photographed and the number of zebrafish with different fluorescence intensities was counted at 48 hpf(Hour post fertilization)of development.Results The successful generation of both transposable plasmids was demonstrated by cellular experiments;hypoxia induced the expression of GFP in Tg(HRE-sv40mp:GFP)but not in Tg(HREM-sv40mp:GFP);both LW6 and Chrysin were able to inhibit GFP expression in Tg(HRE-sv40mp:GFP)zebrafish under hypoxic conditions.Conclusion A transgenic zebrafish model Tg(HRE-sv40mp:GFP)that can respond to hypoxia and be used for HIF-la target drug screening was successfully generated.
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