Rapid Generation of Somatic Mouse Mosaics with Locus-Specific, Stably Integrated Transgenic Elements

Abstract

In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with ''personalized'' driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice.

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter

“…Moreover, the tight regulation of Cre amplification, coupled with the presence of a double-reporter system, allows a strict monitoring of false negatives cells, which are poorly detected in the above-mentioned systems. Recently, a method named MADR has been developed to generate in vivo mosaics exploiting both site-specific recombinase technology and IUE 61 . Although this clever system results in the generation of defined and sparse mosaics similarly to Beatrix, the MADR technique is mainly oriented to generate a gain of function mosaic rather than a KO/WT mosaic on floxed mouse strains for a specific gene of interest.…”

De Novo Generation of Murine and Human MADR Recipient Cell Lines for Locus-Specific, Stable Integration of Transgenic Elements

“…In this protocol, one plasmid expressing both recombinases is used, called pCAG-FlpO-2A-Cre EV ( Figures 2 A and 2C). See Kim et al., 2019 and Rincon Fernandez Pacheco et al, 2020 accompanying STAR protocol for a detailed description and construction of these plasmids. Transfection of MADR pDonor and pCAG-FlpO-2A-Cre EV.…”

“…These cell lines allow the combinatorial use of a wide range of transgenic elements through MADR. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019) .…”

A Genome-wide Library of MADM Mice for Single-Cell Genetic Mosaic Analysis

“…Genetic mosaic animals have been experimentally created in a number of species including C. elegans, Drosophila, and mice among others; and such mosaic analyses provided many fundamental insights in a variety of biological systems (Germani et al 2018;Kim et al 2019;Luo 1999, 2001;Lozano and Behringer 2007;Luo 2007;Rossant and Spence 1998;Xu and Rubin 1993;Yochem and Herman 2003;Zong et al 2005;Zugates and Lee 2004).…”