AAV with GFAP(0.7) promoter driven Cre Inducible C1V1(E162T)-TS-TdTomato

This AAV expresses DIO-C1V1(E162T)-TS-TdTomato driven by an astrocyte GFAP(0.7) promoter.

This 0.7 Kb novel GFAP promoter was constructed by ligating several key regulatory elements of the 2.2Kb human GFAP promoter. It drives essentially the same expression pattern as the parental 2.2 Kb GFAP promoter but with two-fold greater activity.

C1V1 is a chimeric channelrhodopsin made to boost membrane expression higher than VChR1 but with a different light spectrum. It is composed of ChR1 and VChR1 fragments, and implements fast, potent optical excitation at red-shifted wavelengths. C1V1(E162T) mutant has an off-kinetic is half of C1V1.

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.

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).