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H. Yang, H.E. Grossniklaus, G. Fang, X. Huang, W. Gao, P. Liu, X. Mao, I. Schmack; Mouse Melanoma Cells Transfected With EGFP Induced by Lentivirus Stably Grow and Metastasize to Liver in Murine Uveal Melanoma Model . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2214.
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© ARVO (1962-2015); The Authors (2016-present)
To establish mouse melanoma cell lines expressing enhanced green fluorescent protein (EGFP), and to understand whether mouse melanoma cells transfected to express EGFP induced by lentivirus stably grow and metastasize in a murine uveal melanoma model.
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. Mouse melanoma B16LS9, B16F10 cells were seeded in each well of 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. 1×106 cells of B16LS9–EGFP were introduced into C57BL/6 mice by tail vein injection. 5X105cells/2.5ul of B16F10–EGFP were inoculated into the choroid of the right eyes of C57BL/6 mice using a transscleral technique. Control groups were injected by B16LS9, or inoculated with B16F10. The right eyes were enucleated 7 days after inoculation. Hepatic frozen sections were observed under fluorescence microscopy at 10 days after tail vein injection and 21 days after enucleation.
At 24 hours after transfection, EGFP expression was observed under inverted fluorescence microscopy. At 72 hours after transfection, more than 70% of B16LS9, B16F10 expressed EGFP. At 20 days, 3 passages, 70% of B16LS9 and 90% of B16F10, and at 45 days, 6 passages, 90% of B16F10 cells stably expressed EGFP. Histologic examination showed that B16LS9–EGFP and B16F10 cells metastasized to the hepatic tissue in C57BL/6 mice.
Mouse melanoma cells transfected to express EGFP induced by lentivirus stably grow and metastasize in a murine uveal melanoma model. This preliminary study helps establish mouse melanoma cells with overexpressed or silenced specific genes induced by lentivirus that may affect hepatic metastasis.
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