Recombinase-based methods for labeling cells have empowered numerous biological studies of tissue organization, gene expression, and lineage analysis. These strategies commonly employ the cre/lox system in which Cre-mediated recombination removes a loxP-flanked stop codon (lox-stop-lox; LSL) that then allows expression of a downstream reporter, such as a fluorescent protein. While useful, these approaches have some limitations. Firstly, because the recombinase is expressed from only a single gene promoter these schemes typically only label single cell populations that may be too broad from which to collect meaningful data. Further, although labeling narrower cell populations has been achieved by using gene promoters with intersecting expression profiles to drive independently the expression of two different recombinases, even these strategies may not provide the resolution or specificity that is required for a particular experiment. Finally, when these strategies are used for labeling neuron populations, labeling axonal processes with durable fluorescence is challenging, and may compromise interpretation of cellular or tissue architecture. To overcome these challenges, a research team led by Dr. Patricia Jensen at the National Institute of Environmental Health Sciences in North Carolina developed a novel intersectional recombinase reporter allele that allows for labeling more refined and distinct subpopulations of cells. When used to examine neurons in the central noradrenergic system, their reporter mouse was able to label previously inaccessible neuron subtypes with strong axonal fluorescence, allowing detailed analyses of their morphology and axonal projections. This new reporter system may particularly benefit researchers who are studying cell populations that are well-defined genetically by the genes they express but are difficult to resolve and observe morphologically in situ.
To improve on current reporter labeling strategies that employ, at most, two recombinases (Cre and Flp, typically), Dr. Jensen’s team created a new strain that carries a reporter allele that will label cells defined by the overlapping expression of up to three different recombinases, (B6.Cg-Gt(ROSA)26Sortm1.1(CAG-tdTomato,-EGFP)Pjen/J - stock number 026930). This allele, which they term RC::RFLTG, is targeted to the Gt(ROSA)26Sor locus, and contains sequentially: 1.) a rox-flanked stop codon; 2.) an FRT-flanked stop codon; 3.) a loxP-flanked tdTomato –STOP sequence; 4.) eGFP, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), and poly(A) posttranscriptional sequences. In this system, only cells that express both of the two genes whose promoters are driving Dre and Flp recombinase expression are labeled with red fluorescing tdTomato. When these mice are then bred to a mouse expressing Cre recombinase, tdTomato fluorescence switches to eGFP green fluorescence in the cells that also express the gene that drives cre expression. Of note, Flp- or Dre-deleted alleles derived from RC::RFLTG mice (stock numbers 026931 and 026932 respectively), are available also that can be used as double gene reporters.
To demonstrate the utility of their new intersectional reporter allele, the researchers crossed the reporter-bearing mice with recombinase-expressing strains in order to label neural subpopulations in the noradrenergic system. In mice that were quadruple heterozygous for RC::RFLTG, En1dre, DbhFlp, and Gal-cre, eGFP and tdTomato labeled distinct subsets of noradrenergic neurons throughout the locus coerulus. These neural subsets historically have been difficult to label with such specificity, but the intersectional labeling based on the trio of noradrenergic-specific promoters significantly improved their resolution. Further, labeling was observed along the full length of the neurons’ axonal processes, revealing faithfully their organization within the tissue and their projections. The researchers also demonstrated that this labeling approach is compatible with current tissue clearing techniques that render tissues optically transparent, a benefit that may allow for facile three-dimensional reconstruction of cell locations within tissues.
The intersectional labeling approach described by Dr. Jensen’s group has the potential to empower more refined examinations of gene expression, tissue organization, and cell lineages. The further development of additional neuron and cell type-specific dre, FLP, and cre transgenic strains should allow the expansion of this labeling strategy to other neuron and tissue cell types.