Composite mouse Grm6 (mGluR6) regulatory cassette combining shortened intron 4 and intron 3 enhancer sequences with the core 200 bp Grm6 enhancer and a 500 bp proximal Grm6 promoter, giving markedly stronger and more selective AAV-mediated expression in retinal ON bipolar cells than the minimal 200 bp enhancer/SV40 cassette (grm6).
Length: 2197 bp
Strength: strong
Tissue: Retinal ON bipolar cells — rod bipolar cells in mouse, ON cone bipolar cells in marmoset parafovea and far periphery
Assembles a shortened Grm6 intron 4 fragment (~690 bp) and intron 3 fragment (~807 bp) upstream of the 200 bp core Grm6 enhancer and a 500 bp proximal Grm6 promoter (In4s-In3-200En-mGluR500P), totaling ~2.2 kb. By AAV, this cassette produces over 20-fold higher average fluorescence intensity and transduces about 86% of rod bipolar cells in the mouse peripheral retina compared with the minimal 200 bp enhancer/SV40 cassette (grm6); it also targets ON cone bipolar cells in the parafoveal and far-peripheral retina of marmosets (Cronin et al. 2016).
Applications: Used for AAV-mediated delivery of optogenetic actuators and reporters to retinal ON bipolar cells in mouse models of retinal degeneration and in non-human primate (marmoset) retina, where higher transduction efficiency and cell-type selectivity are prioritized over minimizing construct size.
Limitations: At ~2.2 kb, consumes substantially more of the ~4.7 kb AAV packaging capacity than the 0.4 kb grm6 minimal cassette, leaving less room for large effector transgenes such as bulky optogenetic actuators when combined with a polyA signal. The component introns and enhancer must be arranged in the validated In4s-In3-200En-mGluR500P order for full activity (Cronin et al. 2016).
