Fig. 3

Conditional piggyBac transposon mutagenesis substitutes for genomic instability in EGFRvIII-mutant gliomas. a Mouse constructs for piggyBac transposition. The ATP1-S2 transposon line, with 20 copies per cell. Conditional piggyBac transposase targeted to Rosa26 (tissue-specific piggyBac transposase, TSPB), SA = splice acceptor; SD = splice donor; CAG = CAG promoter; SB = Sleeping Beauty; PB = piggyBac inverted repeats; iPBase = insect version of the piggyBac transposase. The transposon can activate gene transcription if it inserts in the same orientation as the gene, usually in a 5′ position. Gene inactivation can occur if the transposon inserts in the body of the gene as a consequence of gene trapping which can occur in either orientation because of the presence of two splice acceptors and bidirectional poly(A) (pA) sites. b Outline of the experimental design: quadruple transgenic mice conditionally activate EGFRvIII expression and piggyBac transposition in the central nervous system. Resultant tumors are examined molecularly by whole-exome sequencing and mapping of transposon insertions. c Histology of EGFRvIII-PB tumors; although not statistically significant, a higher proportion of grade IV brain tumors are observed compared with tumors lacking transposition. d Immunostaining profile of a typical grade III brain glioma from an EGFRvIII-PB mouse, showing strong expression of neural stem and transit-amplifying cell markers. Scale bar corresponds to 2.8 mm for top panel, and 200 μm for all other panels. e Representative karyotype of EGFRvIII-only and EGFRvIII-PB brain tumors, showing polyploidy in the non-PB tumor. f Chromosomal aberrations in EGFRvIII-only and EGFRvIII-PB tumors (n = 3 and n = 5 tumors respectively; mean chromosomal aberrations 19 vs 6.4, p = 0.013, unpaired two-tailed t test; plots show mean ± standard deviation). g Copy number profile of EGFRvIII-PB tumors (n = 20) with focal amplifications and deletions in key genes highlighted. h Mutational profile of 20 EGFRvIII-PB brain and spinal tumors from whole-exome sequencing. i Key cancer genes identified, either as significantly mutated from MuSiC or copy number altered from GISTIC2, across all mouse brain and spinal tumors in both cohorts; each column represent one tumor