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Therefore, when the HIV-1 vector was inoculated into CD4-positive cells, the vector solution was diluted 20-fold to normalize the transduction titer.

Packaging cells in a 100-mm-diameter culture dish were then washed twice in Opti-MEM medium and the combined Lipofectamine™/AP2 vector solution was administered on to the growing packaging 293 cells.

The viral syringe (Neuros Syringe, Hamilton Laboratory Products, Reno, NV) was slowly lowered to depth (approximately 2 mm/min) where 1 μl of viral vector solution was delivered at 0.2 μl/min.

For efficient vector transfection into the human epidermal cell layers, a 1 μg/ml SpRNAi-RGFP vector solution was prepared by mixing 100 μg of the purified SpRNAi-RGFP vector in 1 ml of autoclaved ddH2O with 99 ml of 100% DNase-free glycerin (or glycerol).

For transfection to the human skins in vivo, a pre-made SpRNAi-RGFP vector solution was formed by mixing certain amounts (ie, 1 1000 μg) of the purified SpRNAi-RGFP vector with or without either anti- EGFP, miR-Tyr or miR-Hyal pre-miRNA insert in 1 ml of autoclaved ddH2O with 99 ml of 100% DNase-free glycerin (or glycerol).

To digest unpackaged genomes, the vector solution was resuspended in 240 μl of PBS pH 7.4 1× (GIBCO; Invitrogen S.R.L., Milan, Italy) and then incubated with 1 U/μl of DNase I (Roche, Milan, Italy) in a total volume of 300 μl containing 40 mM TRIS HCl, 10 mM NaCl, 6 mM MgCl2, 1 mM CaCl2 pH 7.9 for 2 h at 37°C.

The integer vector solution is validated using the following "Ratio-Test".

For efficient vector transfection into the human multiple skin cell layers, a 1 μg/ml SpRNAi-RGFP vector solution is made by mixing 100 μg of the purified SpRNAi-RGFP vector in 1 ml of autoclaved ddH2O with 99 ml of 100% DNase-free glycerin (or called glycerol).

The lentiviral and adenoviral vector solutions were microinjected directly into the pancreatic parenchyma at multiple sites.

AAV2/5-VMD2-human Tyrosinase (59) (dose: 2×10e8 GC/eye) was added to the AAV2/8 vector solution that was subretinally delivered to albino Abca4−/− mice (Fig. 5D).

AAV2/1-VMD2 human Tyrosinase (Gargiulo et al, 2009; dose: 2 5 × 10 GC/eye) or AAV2/5-CMV EGFP (encoding normal size EGFP, dose: 4 × 10 GC/eye) were added to the AAV2/8 vector solution that was subretinally delivered to albino mice (Abca4−/−, BALB/c, and sh1; Figs 5B, C and D and 6A and B; supplementary Figs S10 and S14) or pigmented sh1 mice (Fig 8), respectively.