AAV with GFAP(0.7) promoter driven Cre Inducible hChR2(C128T)-mCherry

This AAV expresses DIO-hChR2(C128T)-mCherry driven by an astrocyte GFAP(0.7) promoter.

This 0.7 Kb novel GFAP promoter was constructed by ligating several key regulatory elements of the 2.2Kb human GFAP promoter. It drives essentially the same expression pattern as the parental 2.2 Kb GFAP promoter but with two-fold greater activity.

hChR2 is a humanized version of ChR2 for mammalian expression. Wild-type ChR2, as well as a few of its mutations, provides the fastest excitation of the channelrhodopsins offered, and is widely used in in optogenetics techniques in neuroscience. Mutations at C128 (C128A, C128S, C128T) can extend depolarization up to seconds or even minutes; they are sometimes referred to as step-function opsins or SFOs. hChR2(C128T) mutant provides for a 2-second open-state lifetime. It has showed strong expression in the hippocampal neurons and Pichia pastoris. It becomes inactivated at strong light due to a reaction into a deprotonated species.

In the DIO scenario, the transgene of interest is inserted in reverse orientation relative to the 5' promoter and is flanked by oppositely oriented loxP and lox2272 sites. In the absence of Cre expression, the transgene will not be produced. In the presence of Cre expression, the transgene will be "FLip-EXchanged" or FLEXed, leading to expression of the transgene. This is due to a permanent Cre-mediated recombination/inversion of the flanked transgene. This arrangement is called DIO (double-floxed inverse ORF), Cre-ON, Flex-rev (reverse), Flex-ON/FlexON, or DIO-AAV/AAV-DIO (double-floxed inverse ORF in AAV).