Figure 6: piggyBac transposition in the adult mouse kidney.

Mice received either 50 μg of the piggyBac enhanced firefly luciferase transposon alone (grey triangles) or transposon plus 10 μg of the hyperactive piggyBac transposase (black squares) by renal pelvis injection. The plasmids expressed both enhanced firefly luciferase and hyperactive piggyBac transposase from the following promoters: (a) CMV (transposon alone, n = 4; with transposase, n = 8), (b) EF-1α (transposon alone, n = 5; with transposase, n = 4), (c) γGT1 (transposon alone, n = 4; with transposase, n = 5), and (d) podocin (transposon alone, n = 4; with transposase, n = 5). Either an asterisk (*p < 0.05 by Mann-Whitney U test comparing the area under the curve for each mouse as calculated by the trapezoidal approximation rule) or a pound sign (^#p < 0.05 by Student’s t-test at the last timepoint) indicate statistical significance. (e) Excision assay to detect the molecular junction remaining in the transposon plasmid after the transposon has been removed by active piggyBac transposase. An agarose gel is shown in which lane 1 is the 100 bp ladder and the remaining lanes were each loaded with the same amount of PCR product from a reaction in which the template DNA was either: (2) water, (3) positive control, 4) liver, 5) uninjected kidney, or 6) injected kidney removed from a mouse injected 24 hours prior as in the transposase group in (a). The positive control band is larger than the band in the injected kidney lane because the control was made by digesting the transposon plasmid to remove the majority of transposon sequences and self-ligating the backbone plasmid. This procedure created a DNA template for the PCR reaction that gave a slightly larger PCR product than that produced by direct piggyBac transposon excision.