Figure 1.

Cre-conditional retrograde tracing with PRV-Introvert. A, Schematic of PRV-Introvert construction. Bartha-BAC, the parent of PRV-Introvert, expresses full length PRV tk from its native promoter (i). EGFP (green asterisk) and the sequence for maintenance as a bacterial artificial chromosome (pBluelox; Smith and Enquist, 2000) are inserted into the Us7/Us2 locus. ΔTK has a deletion of the PRV tk gene (ii). In LSL-TK, PRV tk is controlled by a lox-stop-lox cassette between the CMV immediate early (IE) promoter and mCherry-ires-tk, all of which is inserted into the PRV gG/U_S4 locus (iii). In Inverted-tk, mCherry-ires-tk is reversed between two lox sites (iv). In PRV-Introvert, the PRV tk gene is split into 5′ and 3′ ends between two sets of inverted lox sites. Cre recombination flips the 3′ end of PRV tk into the same orientation as the 5′ end and removes two lox sites. The single lox site that remains within the PRV tk transcript is removed by splicing. Open triangles represent loxN sites while lox2722 sites are represented by shaded triangles (v). B, TK activity of parental strain (Bartha-BAC) and mutants. Virus stocks were titered in the presence or absence of AraT, a nucleoside analog that inhibits viral replication in the presence of PRV TK. TK activity is measured as the ratio of the number of plaques without AraT/ with AraT. C, Kaplan–Meier curve showing mouse survival after injection of PRV into the NAc. Bartha-BAC, ΔTK, LSL-TK, Inverted-TK, and Introvert + wt show data for wild-type mice. Introvert + DAT-Cre shows survival of DAT-Cre mice after NAc injection of PRV-Introvert-GFP. Numbers in parentheses are the number of mice injected with each virus. D, PRV-Introvert injected into the NAc of wild-type (1, 3, 5, 7) or DAT-Cre (2, 4, 6, 8). Four days after injection, the brains were perfused, sectioned, and stained with anti-GFP. Scale bars: 250 μm.