Experiment study of tyrosinase gene′s expression in HEK293 cell by MR

Objective To transfect the tyrosinase gene into HEK293 cell as a reporter gene, and to evaluate the tyrosinase gene′s expression by using MRI based on the gene′s property of synthetizing large amount of melanin, and to search a way for evaluating the results of gene expression by MR in vitro.Methods The plasmid of pcDNA3tyr which carried the full-length cDNA of tyrosinase gene was transfected into HEK293 cell by lipofectin, and MR signals of expressed melanin was observed by scanning the transfected cells with MR sequences of T 1WI, T 1WI/SPIR, and T 2WI.Fontana stain and electric microscopy were used to search for melanin granules in transfected cells, and RT-PCR method was used to search for cDNA of tyrosinase gene.Results (1) Plasmids of pcDNA3tyr could be transfected into HEK293 cells and could synthetize a large amount of melanin in them.The synthetic melanin in 106 cells, which had been transfected with 5 μg, 10 μg, and 20 μg plasmids of pcDNA3tyr separately, were all sufficient to be detected by MR and appeared as high signal on MR T 1WI, T 1WI/SPIR, and T 2WI sequences.The more the amounts of transfected plasmids, the higher the signal intensities of MR imaging.On the other hand, 6.25×10 4 cells with 20 μg-plasmid of pcDNA3tyr transfection could also be detected by MR; (2) The melanin granules could be found in HEK293 cells in Fontana stain; (3) The melanin granules and their front bodies could be found in intracytoplasm of HEK293 cell by electric microscopy.(4)The cDNA fragment of tyrosinase gene could be detected in transfected HEK293 cells by RT-PCR.Conclusion The fact that MR could detect the synthetic melanin in HEK293 cells controlled by expression of exogenous gene demonstrated that medical imaging combined with molecular biology technology could evaluate the result of gene expression in vitro, and it also indicated that medical imaging could play an important role in the evaluation of gene therapy following the development of technology.