Inducible Calling Cards: Developing Mouse Reagents for Experimentally Controlled Transposon Insertion in vivo

The piggyBac transposase enables robust forward genetic screens in mice. Further, fusions of piggyBac with specific transcription factors (TFs) enables recovery of recoverable transposons (Calling Cards) for recording location of DNA binding events in vitro and in vivo. Such applications would be enhanced by engineering inducible transposases, such that timing of recording could be precisely controlled in vivo, as has been previously developed in vitro. Here, we tested two approaches for applying inducible Calling Cards (iCC) in the murine brain. We engineered knock-ins of inducible versions of two TFs: Jun, an immediate early gene that serves as a proxy for neural activity, and Sp1, a promiscuous binder of CpG unmethylated regions that indicates active promoters. We fused hyperPiggyBac transposase and a tamoxifen-inducible domain (ERT2) to these TFs and tested the system for efficacy and temporal control of insertions, both in vitro and in vivo. Jun-iCC mice developed normally with no behavioral abnormalities, showed tamoxifen-dependent recording, and captured neural activity during pharmacologically induced seizures. Jun-iCC yields relatively low numbers of insertions, likely due to the transient expression of Jun. In contrast, Sp1-iCC provided substantially higher insertion numbers, but transgenic animals exhibited developmental abnormalities, including reduced viability, anophthalmia, and reduced body weight, suggesting that ERT2 domains may sequester Sp1 and thus significantly impact development. Nonetheless, these inducible Calling Cards mouse lines enable drug-inducible integration of transposon cargo into specific loci in the living mouse.