Triple-cell lineage tracing by a dual reporter on a single allele

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.

Genetic lineage tracing with multiple DNA recombinases: A user's guide for conducting more precise cell fate mapping studies

“…2). For instances, RC::Fela, RC::Frepe, R26-NZG, R26::Flap, R26-TLR, and Rosa26-Confetti2 (47,(54)(55)(56)(57)(58)(59). The structure of some intersectional multicolor reporter systems can be summarized as a Promoter-site1-Stop-site1-site2-reporter A-Stop-site2-reporter B, including RC::Fela, R26-NZG, RC::Frepe or R26::Flap (Fig.…”

“…Therefore, this strategy can be used for consecutively labeling recombinase1 + cell populations by reporter A as well as their intersectional recombinase1 + recombinase2 + subpopulations by reporter B. Furthermore, in R26-TLR, the structure is CAG promoter-site1-Stop-site1-reporter A-CAG promoter-site2-Stop-site2-reporter B (57). The expression levels of reporter A and reporter B are driven independently by two recombination events, which do not interfere with the readouts with each other.…”

Bronchioalveolar stem cells in lung repair, regeneration and disease

“…Although there are benefits to the indirect methods of detecting and quantifying BASCs, several recent studies have described new genetically modified mice that allow for in vivo quantification, tracing, and functional analysis of BASCs (Table 1) [25,26,29,30]. Salwig et al developed a genetic approach allowing specific labeling and manipulation of BASCs, based on the co-expression of CCSP and SP-C [25].…”

“…Multicolor reporters are a powerful platform for simultaneously tracking cell populations in vivo. [30].…”

Cardiac Fibrosis and Cardiac Fibroblast Lineage-Tracing: Recent Advances

“…More important, due to the high specificity of Cre-loxP and Dre-rox systems, the dual lineage-tracing combining these two systems is a recent trend in lineage-tracing studies. Mice carrying both Cre-loxP and Dre-rox permit the simultaneous tracing of two distinct or related cell types (Liu et al, 2019). These mice may be particularly useful in the study of cardiac fibroblast differentiation and transdifferentiation when the precursor cells and their descendants express different marker genes.…”