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Infection of RIP-Tag; RIP-tva Pancreatic Islet Cells In Vitro and In Vivo

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posted on 2007-10-16, 01:13 authored by Yi-Chieh Nancy Du, Brian C Lewis, Douglas Hanahan, Harold Varmus

(A) Experimental strategy. RIP-Tag transgenic mice were crossed with RIP-tva transgenic mice to generate RIP-Tag; RIP-tva bitransgenic mice. For in vitro experiments, tumors from 16-wk-old bitransgenic mice were isolated and cultured in vitro, and tumor cells were infected with RCASBP viruses (left). For in vivo infection, RCASBP viruses were delivered into 7-wk-old bitransgenic animals through intra-cardiac (I.C.) injection. Characterization of islet cells/tumors was performed 2, 5, or 9 wk after viral delivery (right).

(B and C) Example of infection in vitro with an RCASBP vector. Bright-field image of RIP-Tag; RIP-tva tumor cells infected with RCASBP-GFP in vitro (B) and fluorescent image showing GFP expression (C) were taken 1 wk after infection. The images are representative of more than 20 fields from two independent infections.

(D) Flow cytometry plots of islet cells from wide-type mouse (C57BL/6) injected with RCASBP-GFP (upper left), RIP-tva transgenic mouse injected with RCASBP-GFP (upper right), and RIP-Tag; RIP-tva bitransgenic mice injected with RCASBP-ALPP (lower left) or RCASBP-GFP (lower right) via intra-cardiac injection. RCASBP viral supernatants were delivered at the age of 7 wk. Two weeks later, mice were sacrificed, and pancreases were digested into single-cell suspension. Cells were analyzed for TVA+ and GFP by FACS. The percentage of cells in each gated panel is indicated in the corners. Approximately 11%–18% of pancreatic cells isolated from RIP-tva or RIP-Tag; RIP-tva bitransgenic mice by this method were TVA+ (upper quadrants), and about 20% of TVA+ cells from bitransgenic animals infected with RCASBP-GFP expressed GFP.