Transfecting pcDNA 3.0-tyr into GM Cells and Identification

Objective To study the gene expression of pcDNA3.0-tyr in GM cells(GM0639) from the skin of the normal subject in vitro.Methods pcDNA3.0-tyr eukaryotic expression plasmids were multiplied and purified,and its integrity was determined by means of sequencing and restriction analysis.The plasmids were transfected into GM cells by electroporation.Then reverse transcription PCR was performed in transfected cells.The tyrosinase gene expression and its levels in transfected cells were confirmed by measuring absorption spectrum at 490 nm and staining with the Fontana stain.Results The result of sequencing and restriction analysis confirmed that the eukurytoic expression vector induced contains the full-length human tyrosinase cDNA.Reverse transcription PCR product of transfected cells showed a band that coincided with that of reverse primers,whereas the non-transfected cells don't show this band.The value of A490 in transfected cells is greater than that in mock-transfected and non-transfected cells(P0.001).Fontana stain showed that the brownish color granules in pcDNA3.0-tyr transfected cells,which represented induced melanin,and the absence of these in non-transfected cells.Conclusion The plasmids of pcDNA3.0-tyr can be transfected into human GM0639 cells and synthesize melanin.