Figure 2.

PRV-Introvert-GFP does not express GFP or spread in the CNS without Cre recombinase. A, Cre-dependent expression of PRV tk and GFP using a FLEX intron and 2A ribosomal skip sequence. Exposure to Cre inverts the 3′ exon of PRV tk (TK-2) and the 5′ exon of GFP (GFP-1) and excises one loxN (open triangles) and one lox2722 site (shaded triangles). The remaining lox sites are removed by splicing. B, Cre recombinase is required for GFP expression during PRV-Introvert-GFP infection of PK15 cells. C, PRV-Introvert-GFP injection of wild-type and DAT-Cre mice. 1, Wild-type mouse showing limited expression of viral proteins and no GFP three days after NAc injection with PRV-Introvert-GFP. 2, Representative sections from DAT-Cre mouse four days post NAc injection. ac: anterior commissure. D, Time course of PRV spread in DAT-Cre 24, 48, 72, and 96 h after bilateral injection in the NAc. Anti-TH staining marks dopamine neurons. E, Structures infected 48, 72, and 96 h after infection that represent second or higher order connections to DAT-Cre starter cells in the VTA. Scale bars: B, 100 μm; C, 250 μm; D, E, 500 μm.