We used a virus co-expressing Cre recombinase and the red fluorescent protein tdTomato using Thosea asigna virus 2A self-cleavage sequences (AAV8/iCre-2A-tdTomato) to test the accuracy with which a virally-expressed fluorescent protein labeled cells that underwent Cre-mediated recombination. The AAV8-iCre-2A-tdTomato virus was co-injected at varying titers with AAV8-YFP into P0 pups from the R26R lacZ reporter line

(Soriano, 1999). Importantly, we found that tdTomato (Figs 9A–C) and β-gal (Figs 9G–I) were reliably co-localised (Figs 9P–R), indicating that the viral fluorescent protein served as an Roxadustat accurate indicator of Cre-induced genetic modification within most brain regions, including the hippocampus and striatum (96 ± 0.4% of cells in the CA1 hippocampus and 94 ± 0.6% of cells in the striatum co-expressed tdTomato and β-gal; n = 22–25 sections, four to five sections/brain from five animals). Co-localisation was less dependable within the neocortex where, in layers 2/3 and 4, β-gal was often detected in cells with very low red fluorescence (89.4 ± 0.1.0% of cells in the cortex

co-expressed tdTomato and β-gal, n = 29 sections, six sections/brain from five animals). Although the majority of controllable genetic models are based on the Cre-loxP system, transgenic mice that utilise the tTA-rtTA system for inducible, reversible expression of transgenes are becoming increasingly available. To test viral delivery of tTA, we designed a virus encoding tdTomato-2A-tTA (AAV8-tdTomato-2A-tTA) and injected it into green fluorescent protein (GFP) tet reporter Alpelisib purchase mice (tetO-nls-GFP-LacZ) (Mayford et al., 1996). We observed reliable co-expression of viral tdTomato and transgenic GFP in most brain areas in these mice (Fig. 10). Neonatal injection of 5.0 × 109 particles Pregnenolone of AAV8-tdTomato-2A-tTA

into GFP tet reporter mice resulted in highly reliable co-expression of tdTomato and GFP in individual neurons, with very low mismatch in the hippocampus and cerebellum and slightly higher mismatch in the cortex (98 ± 0.5% of pyramidal neurons in CA1, 95 ± 1.0% of Purkinje cells in the cerebellum, and 83 ± 2.1% of neurons in the cerebral cortex co-expressed tdTomato and GFP; n = 12–19 sections, three to four sections/brain from four to five animals). These data provide proof-of-principle that the neonatal injection of viruses co-expressing a transgene and fluorescent marker can be employed to genetically manipulate a subset of cells in brain tissue and accurately identify these cells for further study. We recognised that the distributed transduction pattern of our low-titer injections resembled the sparse labeling seen in the Thy1-GFP transgenic M and S mouse lines that have been widely used for imaging dendritic processes in vivo (Holtmaat & Svoboda, 2009; Holtmaat et al., 2009). We wondered if viral transgenesis might allow live imaging of neurons in the intact brain as had these two Thy1 transgenic lines.