Abstract
Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter (R26-TLR), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre+Dre-, Cre-Dre+, and Cre+Dre+ cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity.
Highlights
Genetic lineage tracing is widely used to study organ development and tissue regeneration
Multiple different epithelial cells in the distal airway are derived from club cells or alveolar type 2 cells and bronchioalveolar stem cells (BASCs)2 after lung injury [15, 16]
Our new mouse reporter Rosa26 traffic light reporter (R26-TLR) extents the scope of cell type labeling with one reporter allele, which could be broadly used for diverse cell origin and cell fate studies in development, disease, and regeneration
Summary
The Cre-loxP system is a widely used site-specific recombinase-based system. Like the Cre-loxP system, Dre recombinase targets its recombination site rox [19]. Dre and Cre recombinases driven by two different promoters recombine rox or loxP sites, resulting in ZsGreen or tdTomato reporter expression, respectively. When the two promoters are both active in a cell, the cell expresses ZsGreen and tdTomato at the same time, yielding yellow fluorescence (Fig. 1, B and C) We named this reporter line Rosa traffic light reporter (R26-TLR). All cells were ZsGreenϩtdTomatoϪ in the CAG-Dre;R26-TLR line, whereas all cells were ZsGreenϪtdTomatoϩ in the ACTBCre;R26-TLR line, indicating that Dre or Cre recombinase recombined its own target site (Fig. 1E). In the CAGDre;ACTB-Cre;R26-TLR triple-positive line, all cells expressed both ZsGreen and tdTomato, yielding yellow fluorescence in those DreϩCreϩ cells. Taken together, these data demonstrated that the new reporter R26-TLR could be used for tracking different cell populations simultaneously in vivo
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