Fig. 7: Hermes transposition assay in HEK293T cells.

a Schematic of the experimental procedure. The plasmid p-helper encoding the Hermes transposase (tnp) and the p-donor containing the puromycin resistance gene puro^R (1.5 kb) flanked by the Hermes transposon left-end (LE) and right-end (RE) are transfected into HEK293T cells. The cells that experience a chromosomal integration of the LE-puro^R-RE transposon are selected against puromycin and the colonies are counted. b–e Top: Schematic of the Hermes LE, RE, and their variants (truncation or mutations) used in the assay. The terminal inverted repeats (TIRs) are represented by blue arrowheads and the subterminal repeats putative Hermes BED domain binding sites are depicted as red bars. Bottom: Histograms of the colony count for each p-donor. The combination of the transposon ends is reported on the x-axis for each p-donor. Each black circle represents one independent data point. The mean values are plotted as columns (red-stripped and black-stripped columns for the controls and white columns for the experimental data) the standard deviations are represented with bars. In b n = 8 (experimental data and LE/RE) or n = 4 (pUC19 controls) or n = 3 (pB). In c–e n = 4 (experimental data) or n = 3 (controls). Positive controls (red-stripped columns) n = 8 in b, c, n = 3 in d, e. pUC19 controls n = 4 (in b) or 3 (in c–e). The statistical two-tailed unpaired t-test was applied to determine whether the experimental data were statistically different from the LE/RE (in b, c, e) or LE223/RE (in d) experiment (annotated with a red star when significant or a red “ns” when not) and their pUC19 controls (annotated with a black star when significant or a black “ns” when not), respectively. In b *p < 0.0001 and ^nsp = 0.2478. In c ^red*p < 0.0001 except for LE190/LE ^red*p = 0.0096, ^{red ns}p = 0.2357; 0.5290; 0.6032, and 0.9953 (from left to right, respectively), ^black*p < 0.0001 except for LE306/LE ^black*p = 0.0002, ^{black ns}p = 0.3997; 0.6441; 0.1525; 0.4557 (from left to right, respectively). In d ^red*p < 0.0001 except for RE215/LE223 ^red*p = 0.0047, ^{red ns}p = 0.2549, ^black*p < 0.0001, ^{black ns}p = 0.0036 and 0.0066 (from left to right, respectively). In e *p < 0.0001 except for LEmut2 ^red*p = 0.0002, LEScr50 ^black*p = 0.0036 and LEScr140 ^black*p = 0.0082. The piggyBac transposition system (pB) was used as a general control of the experimental design.