FIGURE 2.

Gene delivery to deep brain neuron by single-cell electroporation. (A) Electroporation efficiency under different conditions. Percentage of GFP-expressing sites per penetration site (1 penetration site per target area) is shown; in the case of multiple-site injection, the percentage of GFP-expressing areas per target area (3 penetration tracts per target area) is shown. Total number of penetration sites (or total number of target areas for multiple-site injection) is given above each bar. + pressure, electroporation under weak pressure; DNA injection, electroporation after injecting DNA under strong pressure; R, electroporation by monitoring tip and cleft resistance. (B) The number of GFP-expressing cells per GFP-positive site is shown; in the case of multiple-site injection, the number of GFP-expressing cells per GFP-positive target region is shown. The box color (white to black) indicates the number of GFP-expressing cells. Total number of GFP-positive sites (or total number of GFP-positive regions for multiple-site injection) is given above each bar. (C) A sharp glass electrode with a 5-mm length of shank and 50-μm tip diameter prepared by cutting the glass tip. (D,E) GFP-expressing cells 2 days after electroporation. Most of transfected cells showed intense green fluorescence (D), but we found damaged cells (arrowheads in panel E) with weak GFP and poor processes near the penetration sites (asterisks). The penetration sites exhibited green and red autofluorescence. (F) Whole brain slice including two penetration tracts (arrows) and GFP-expressing cells (arrowheads) at both sides. Autofluorescence was detected along the penetration tracts (2 and 3, higher magnification view of areas indicated by arrows; 1, view of a serial section at a 100 μm distance from panel 2, 3’, higher magnification view of areas indicated in panel 3 with green and red fluorescence) (G) Distance of GFP-expressing cells from the penetration tracts. GFP-expressing cells were detected far from penetration sites after strong pressure DNA injection than after weak air pressure (“+ pressure”) (p = 0.0115, t-test with Welch’s correction; p < 0.05, χ²-test). (H) Relationship between tip and cleft resistance (R) and depth of the electrode from the pia mater. As resistance increased, square pulses were delivered at the points indicated by the lightning shape. Scale bars, 1 mm for panel (F); 100 μm for panels (D,E,F1–3’).