AAV with CMV7 promoter driven Cre Inducible eArch3.0-mCherry

This AAV expresses DIO-eArch3.0-mCherry driven by an ubiquitous CMV7 promoter.

The CMV7 promoter is about 0.35 kb in length and is a shortened version of the commonly used CMV promoter. CMV7 has a truncated version of the CMV enhancer. When tested in several cell lines, this promoter provides an expression level that is about 3-5 fold lower than that of the original 0.6Kb CMV promoter.

Arch and ArchT are light-driven proton pumps found in archaebacteria. They provide silencing via hyperpolarization. Only slightly slower than NpHR2, their peak activation falls at 566 nm. ArchT has about a three-fold higher light sensitivity compared to Arch leading to a similar level of inhibition but increased volume of tissue that will be inhibited.

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).