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H.E. Grossniklaus, H. Yang, W. Gao, P. Liu, G. Fang, X. Huang, J. Yu, C.–L. Hsieh; Human Uveal Melanoma Cells Transfected With Egfp Induced by Lentivirus Stably Grow in vitro And Metastasize in vivo . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2219.
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© ARVO (1962-2015); The Authors (2016-present)
To establish a human uveal melanoma cell line expressing enhanced green fluorescent protein (EGFP), and to understand whether human uveal melanoma cells transfected to express EGFP induced by lentivirus stably grow in vitro and metastasize in vivo.
Human uveal melanoma Mel290 cells were seeded in each well of a 6–well–plate and incubated. When cells reached 30–50% confluence, 1×106 infectious units(I.U.) of lentiviral EGFP expression vector were added. Lentivirus was added to control wells. For lentiviral vector construction, the lentiviral backbone used in these experiments is based on FUGW. Vector particles were produced in 293FT cells by transient co–transfection the transfer vector, the HIV–1 packaging vector ΔR8.9, and the VSVG envelope glycoprotein into 293T cells. Approximately 72 hours after transfection, virus–containing supernatant was removed, filtered and tittered. 1×106 cells of Mel290–EGFP were introduced into NU/NU mice by tail vein injection. Control groups were injected with Mel290. Hepatic frozen sections were observed under fluorescence microscopy at 10 days after tail vein injection.
At 24 hours after transfection, EGFP expression was observed under inverted fluorescence microscopy. At 72 hours after transfection, more than 70% of Mel 290 cells expressed EGFP. At 20 days, 3 passages, Mel 290 cells stably expressed EGFP. At 45 days, 6 passages, 90% of Mel 290 cells stably expressed EGFP. Histologic examination showed that Mel 290–EGFP cells metastasized to the NU/NU mice livers.
Human uveal melanoma cells transfected to express EGFP induced by lentivirus stably grow in vitro and metastasize to mouse hepatic tissue. This preliminary study helps establish human melanoma cells with overexpressed or silenced specific genes induced by lentivirus that may affect hepatic metastasis.
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