AAV with CAG2 promoter driven Cre Inducible eNpHR3.0-EGFP

This AAV expresses DIO-eNpHR3.0-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.

NpHR is a halorhodopsin or a light-sensitive chloride channel that provides silencing of neuronal activity by hyperpolarization. It offers fast inhibition, and its peak activation falls at 589 nm.

The trafficking signal (KSRITSEGEYIPLDQIDINV) is also from the inward rectifier potassium channel Kir2.1; it serves to dramatically reduce intracellular accumulation and improve membrane targeting, leading to a profound increase of photocurrents. This is the basis for many third-generation optogenetics tools.

This ER export motif (FCYENEV) is from the vertebrate inward rectifier potassium channel Kir2.1. The motif prevents aggregate formation, decreases intracellular accumulations, and enhances tolerability at high expression levels. This is the strategy for developing many second-generation optogenetics tools.

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).