piggyBac Transposon Somatic Mutagenesis with an Activated Reporter and Tracker (PB-SMART) for Genetic Screens in Mice
Figure 3
Simultaneously inducing mutations and tracking mutated cells with a PB transposon system.
(A) PB[mut-RFP] couples RFP expression with ectopic expression of a downstream gene or partial gene transcript via the IRES (brown box). CS (purple box) prevents unwanted RFP expression from transgene concatamers. (B) PB[mut-RFP] mobilization in HEK 293 cells induces RFP-positive clones that colocalize with myc-tagged proteins ectopically expressed by the mutator transposon. (C) Transgenic lines carrying two to 200 copies of PB[mut-RFP] were generated. (D) Significant PB transposition-induced embryonic lethality was observed when high-copy lines were crossed to Act-PBase. (E) Low-copy PB[mut-RFP] lines crossed to Act-PBase mobilized the mutator transposon as determined by PCR with primers directed against sequences flanking the transposon. A 212-bp excision product was specifically detected in double transgenics but not in single transgenic littermates. A 700-bp unexcised product was observed in all PB[mut-RFP] mice. (F) Shown here in the dorsal skin of a PB[mut-RFP]2a;Act-PBase mouse, RFP-positive foci can be detected ex vivo over background signal. Units, photons·s−1·sr−1·ìM−1. (G) RFP-marked tumor formation (arrowhead) can be visualized ex vivo as seen in the upper panels showing a PB[mut-RFP]2b;Act-PBase mouse. In the lower panels, a stomach tumor dissected from a PB[mut-RFP]7;Act-PBase mouse is strongly RFP-positive. Units, photons·s−1·sr−1·µM−1.