AAV with CamKII promoter driven Cre Inducible eMac3.0-EGFP

This AAV expresses DIO-eMac3.0-EGFP driven by a neuron CamKII promoter.

This 1.3 Kb promoter is derived from the murine α-Calcium/calmodulin-dependent kinase II (CaMKII), a gene with expression restricted to excitatory neurons in the neocortex and hippocampus. In cortical neurons, it was shown that the promoter activity of CaMKII(1.3) is about double that of CaMKII(2.4), probably due to some inhibitory element(s).

Mac is a light-activated proton pump from a species of fungus. Its peak activation is at 540 nm. It provides neuronal silencing under blue-green light, enabling two different cell populations to be silenced when used in combination with another silencing protein.

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).