Figure 3

Low-leak Cre-dependent expression using the doublefloxed inverted open-reading-frame (DIO) strategy. The combination of a transgenic mouse expressing Cre recombinase in specific neuronal subtypes and the injection of a virally encoded DIO opsin (a) results in the physical inversion of the open reading frame (ORF) in only that population (b,c), which may be transient and revert back to the original state or undergo further recombination to be permanently anchored in the sense direction, resulting in functional expression of the opsin (c). The DIO strategy may be contrasted with the lox-stop-lox (“floxed STOP”) strategy (d,e). In the absence of Cre recombinase, lox-stop-lox (d) allows for some level of expression leak as assayed by both enhanced yellow fluorescent protein (eYFP) expression and fluorescence-activated cell sorting (FACS) analysis. Because the ORF of an opsin in DIO configuration encodes nonsense (e), there is no functional expression in the absence of Cre recombinase. Adapted with permission from Sohal et al. (2009) and F. Zhang and K. Deisseroth.