Figure 4. AAV-PHP.eB and AAV-PHP.S transfer multiple genomes per cell and enable tunable multicolor labeling.

(a) Co-administration of a cocktail of three ssAAV vectors that each express one of three XFPs (top, schematic) from the hSyn1 promoter labels individual cells with a range of hues. Representative images of the cortex (left, AAV-PHP.eB; total dose: 4 × 10¹¹ vg) and the enteric nervous system (right, AAV-PHP.S; total dose: 1 × 10¹² vg). The inset highlights the dense network of neurites labeled with a range of hues. Schematics show one-component (b) and tTA-TRE-based two-component systems (c). Expression from the TRE-XFP vectors is dependent on co-transduction with a tTA (tet-off) inducer vector that contains a positive feedback loop to increase tTA expression (ssAAV-PHP.eB:ihSyn-tTA). (d) Representative images using the one-component expression system with a 3XFP cocktail expressed from the CAG promoter at an equal dose per XFP (high: total dose = 2 × 10¹¹ vg; low: total dose = 1 × 10¹⁰ vg). (e) Representative images using the two-component system: 1 × 10¹² total of the TRE-3XFP cocktail and a high (1 × 10¹¹ vg) or low (1 × 10¹⁰ vg) dose of the tTA inducer. (f) Percentages of individual cells that express one, two, or all three XFPs. (g) MIP (left) of a coronal section from the olfactory bulb of a Tbx21-Cre mouse transduced with a cocktail of ssAAV-PHP.eB:TRE-DIO-3XFP (1 × 10¹² vg total dose) and sAAV-PHP.eB:ihSyn-tTA (1 × 10¹⁰ vg). The morphologies of local neuronal dendritic arbors were traced with NeuTube and imported into Imaris v8.3 for 3D rendering (middle) and overlay (right). Scale bars (d and e) are 50 µm and are MIPs of 40 µm z-stacks. The total volume in (g) is 0.042mm³. All images were acquired on tissue extracted three weeks after intravenous AAV delivery in adult mice, except for (a) where the expression was assessed after 11 days.