Figure 1: Hyperactive piggyBac transposase (sPBo) paired with 80 V pulses results in transgene expression in a large portion of the targeted area in zebra finch hatchlings.

(a) Plasmid constructs used in this study. Donor pPB plasmids contain inverted terminal repeats (ITRs) surrounding the transgene expression cassettes, each containing a promoter (CAG or human Synapsin 1 [hSyn1] promoters) and fluorescent protein transgenes (eGFP, mRFP, or CFP). (b) Schematic of plasmid infusion targeting and paddle placements. Left panel: Infusions were targeted along a 45° angle visualized from the midline and Y₀ (the anterior-most boundary of the cerebellum). Middle panel: Infusion into the chick’s left ventricle (arrow). Fast green dye in the plasmid mixture allows visual confirmation that the plasmid solution has filled the ventricle (arrow). Right panel: Schematic showing tri-electrode paddle positioning, as seen from the top and back views of the chick’s head. Positively-charged paddles (red) are placed on either side of the head, in line with the posterior-most portion of the filled lateral ventricles. The negatively-charged paddle is placed on top of the head, directly over the posterior-most portions of the lateral ventricles. (c) Quantitative comparison of the effects of various electroporation conditions on the percent of the targeted area containing fluorescent protein-positive (FP+) cells. sPBo is significantly more effective than wildtype (WT) piggyBac at all voltages tested (*p < 0.05; vs. 70, 80, 90 V sPBo). sPBo paired with 100 ms 80 V pulses delivered at an interpulse interval of 900 ms using 3 mm-wide paddles was significantly more efficient than all other conditions (^#p < 0.001). n = 2–3 per group. Bar graphs indicate the group mean ± SEM; ○ = individual birds. (d) Representative sagittal plane images of fluorescent protein-positive (FP+) cells and DAPI-stained cell nuclei along the lateral ventricles 48 h after in vivo electroporation with sPBo, 3 mm-wide paddles, and 100 ms 80 V pulses delivered with an inter-pulse interval of 900 ms. White boxes outline areas magnified in the panels directly above. Scale bars = 100 μm. (e) Summary of the survival, mortality, and efficacy percentages obtained over the course of this study.