Generatingin vivosomatic mouse mosaics with locus-specific, stably-integrated transgenic elements

Abstract

Viral vectors and electroporation (EP)-mediated gene transfers are efficient means of inducing somatic mosaicismin mice, but they lack the exquisite control over transgene copy number, gene zygosity, and genomic-locus specificity that genetically engineered mouse models (GEMMs) provide. Here, we develop and demonstrate a simple and generalizable in vivo method, mosaic analysis by dual recombinase-mediated cassette exchange (MADR). MADR allows for stable labeling of mutant cells express transgenic elements from a precisely-defined chromosomal locus. To test our method, we generated reporter-labeled lineages from stem and progenitor cells in a well-defined Rosa26 mTmG mouse. We demonstrate the power and versatility of MADR by creating novel glioma models with mixed, reporter-defined zygosity or with "personalized" driver mutations from pediatric glioma-each manipulation altering the profile of resulting tumors. Thus, MADR provides a high-throughput genetic platform for the dissection of development and disease, and this rapid method can be applied to the thousands of existing gene-trap mice.

Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells

“…Toward this end, targeting the AAVS1 safe landing may allow for long-term stable integration and methylation-resistant expression of transgenes ( Hatada et al., 2015 , Oceguera-Yanez et al., 2016 ). Importantly, we have observed marked dox-mediated transgene expression at the single-copy level using the same TRE-Bi promoter used in this study ( Kim et al., 2016 ). Finally, a limitation to translate this strategy into humans may be the large area of the brain that needs to be covered for a therapeutic effect.…”

Organoid and Organ-on-a-Chip Systems: New Paradigms for Modeling Neurological and Gastrointestinal Disease

“…The use of postnatal electroporation for modeling pediatric glioma allows for the rapid and stable insertion of oncogenic drivers to stem and progenitor cells in the murine brain [55]. Combining this technique with dual recombinase-mediated cassette exchange allows for site-specific insertion of oncogenic drivers at the single copy level, adding to the physiological mimicry of patient glioma [56]. While all of the abovementioned techniques lend to a better understanding of pathophysiological tumor progression in mice, the differences pertaining to human vs mouse neural development speak to the need of human-specific models.…”