iFlpMosaic enable the multispectral barcoding and high-throughput comparative analysis of mutant and wildtype cells.

In order to understand gene function, cells mutated for a gene need to be compared with normal cells. In most biomedical studies this comparative analysis is carried out with cells present in different animals and therefore not experiencing the same microenvironment or epigenetic changes. Here we present a large set of new genetic tools and mouse lines, that enable the Flp recombinase-dependent ratiometric induction and single cell clonal tracking of multiple fluorescently labelled normal and Cre-mutant cells, from distinct or the same progenitor cells. The labelled cells can be profiled in situ by multispectral imaging, or by FACS and scRNA-seq. With these new tools, normal and mutant cells can be ratiometrically induced and multispectrally barcoded within the same temporal window and tissue microenvironment. This enables a better understanding of how induced genetic mutations affect the biology of single cells with higher accuracy and reliability during tissue development, homeostasis, or disease. Overall design: For RNA-seq analysis, embryos were dissociated and Tomato-KO or YFP-WT cells were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of Tomato signal and YFP signal. Then both cell populations were analyzed using scRNAseq.