AAV with CAG2 promoter driven Cre Inducible hChR2(C128T)-EGFP

This AAV expresses DIO-hChR2(C128T)-EGFP driven by an ubiquitous CAG2 promoter.

The CAG2 promoter is derived from the commonly used 1.8kb CAG/CBA (also called CAGGS) promoter. To increase the cloning capacity of size-limiting vectors using the CAG promoter, such as AAV or lentiviral vectors, CAG2 was developed by deleting a portion of about 0.6Kb of the chicken beta-actin intron from the original CAG promoter.

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).