AAV with CAG2 promoter driven Cre Inducible hChR2(H134R)-mCherry

This AAV expresses DIO-hChR2(H134R)-mCherry 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.

Channelrhodopsins are light-gated ion channels that exist naturally in microalgae. hChR2 is a humanized version of ChR2 for mammalian expression. It is maximally excited by 470 nm light. The wild-type, as well as a few mutations, provide the fastest excitation of the channelrhodopsins offered, and are widely used in optogenetics techniques in neuroscience. Due to delayed channel closure, hChR2(H134R) is a gain-of-function mutation that produces larger photocurrents than wild-type hChR2, but slows down channel kinetics.

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).