Dual Cre and Dre recombinases mediate synchronized lineage tracing and cell subset ablation in vivo

Abstract

Genetic technology using site-specific recombinases, such as the Cre-loxP system, has been widely employed for labeling specific cell populations and for studying their functions in vivo. To enhance the precision of cell lineage tracing and functional study, a similar site-specific recombinase system termed Dre-rox has been recently used in combination with Cre-loxP. To enable more specific cell lineage tracing and ablation through dual recombinase activity, we generated two mouse lines that render Dre- or Dre+Cre-mediated recombination to excise a stop codon sequence that prevents the expression of diphtheria toxin receptor (DTR) knocked into the ubiquitously expressed and safe Rosa26 locus. Using different Dre- and Cre-expressing mouse lines, we showed that the surrogate gene reporters tdTomato and DTR were simultaneously expressed in target cells and in their descendants, and we observed efficient ablation of tdTomato⁺ cells after diphtheria toxin administration. These mouse lines were used to simultaneously trace and deplete the target cells of interest through the inducible expression of a reporter and DTR using dual Cre and Dre recombinases, allowing a more precise and efficient study of the role of specific cell subsets within a heterogeneous population in pathophysiological conditions in vivo.

Keywords: Cre-loxP; DTR; Dre-rox; cell ablation; dual recombinases; lineage tracing.

Figure 1

Generation of the R26-R-tdT-DTR mouse line.A, a schematic figure showing the gene targeting strategy for generation of R26-R-tdT-DTR allele. B, a cartoon figure showing expression of tdTomato and DTR after Dre-rox recombination–mediated removal of Stop cassette. C, a cartoon figure showing tissue-specific expression of DTR and DT-induced cell ablation. D, genotyping verification of R26-R-tdT-DTR mice by PCR. E, immunostaining for tdTomato shows no expression of tdTomato in R26-R-tdT-DTR mice. The scale bar represents 100 μm. Each image is representative of five individual biological samples. DTR, diphtheria toxin receptor; DT, diphtheria toxin.

Figure 2

Expression of DTR and tdTomato in R26-R-tdT-DTR induced by Cdh5-Dre–mediated recombination.A, a cartoon figure showing the mating strategy of mice. B, a schematic figure showing the genetic recombination of the R26-R-tdT-DTR allele with Cdh5-Dre. C, a schematic figure showing the experimental design. D, whole-mount epifluorescence views of multiple organs collected from Cdh5-Dre;R26-R-tdT-DTR mice. Yellow scale bar represents 2000 μm. E, immunostaining for DTR, CD31, and tdTomato on sections of Cdh5-Dre;R26-R-tdT-DTR mice shows tdTomato and DTR expression in multiple tissues or organs. White scale bar represents100 μm. F, a schematic figure showing experimental strategy. G, whole-mount view of small intestine after DT or PBS treatment. Yellow scale bar represents 2000 μm. H, survival of DT treatment- and control-mice. I, immunostaining for tdTomato, DTR, and CD31 on intestine sections after DT or PBS treatment. White scale bar represents 100 μm. Each image is representative of five individual biological samples. DTR, diphtheria toxin receptor; DT, diphtheria toxin.

Figure 3

Generation of the R26-LR-tdT-DTR mouse line.A, a schematic figure showing the gene targeting strategy for the construction of the R26-LR-tdT-DTR allele. B and C, cartoon figures showing tdTomato and DTR expression after both Cre-loxP– and Dre-rox– mediated recombinations. D, a cartoon figure showing tissue-specific expression of DTR and genetic ablation of cells after DT treatment. E, immunostaining for tdTomato on sections of multiple tissues or organs collected from ACTB-Cre;R26-LR-tdT-DTR mice (upper panel) or CAG-Dre;R26-LR-tdT-DTR mice (lower panel). The scale bar represents 100 μm. Each image is representative of five individual biological samples. DTR, diphtheria toxin receptor; DT, diphtheria toxin.

Figure 4

DTR-mediated cardiomyocyte ablation after DT administration.A, a cartoon figure showing the mating strategy for the generation of ACTB-Cre;Tnni3-Dre;R26-LR-tdT-DTR triple transgenic mice. B, a schematic figure showing the genetic recombination of the R26-LR-tdT-DTR allele with Tnni3-Dre and ACTB-Cre. C, a schematic figure showing experimental design. D, a water maze experiment for movement tracking of mice. E, echocardiography of mice treated with DT or PBS. F, a cartoon figure indicating heart injury and the inactive state of mice after DT treatment. G, whole-mount view of triple transgenic mice treated with PBS or DT. Yellow scale bar represents 2000 μm. H, immunostaining for DTR and tdTomato on heart sections of triple transgenic mice. White scale bar represents100 μm. I, immunostaining for cTnT and tdTomato on heart sections of triple transgenic mice. White scale bar represents 100 μm. J, immunostaining for Tunel and tdTomato on heart sections. White scale bar represents 100 μm. Each image is representative of five individual biological samples. DTR, diphtheria toxin receptor; DT, diphtheria toxin.

Figure 5

DTR-mediated specific ablation of Cyp2e1⁺peri-central hepatocytes.A, a cartoon figure showing the mating strategy for generation of Cyp2e1-DreER;R26-LR-tdT-DTR double transgenic mice. B, a schematic figure showing the genetic recombinations of the R26-LR-tdT-DTR allele with Cyp2e1-DreER and AAV8-TBG-Cre. C, a schematic figure showing experimental design. D, whole-mount epifluorescence view of livers collected from mice treated with DT or PBS. Yellow scale bar represents 2000 μm. E and F, immunostaining for tdTomato, HNF4a (E), or DTR (F) on liver sections treated with PBS. White scale bar represents 100 μm. C, central vein. G, immunostaining for CYP2E1, tdTomato, and E-cad on liver sections from mice treated with PBS or DT. White scale bar represents 100 μm. H, quantification of the percentage of tdTomato⁺ hepatocytes (DT group) in comparison of tdTomato⁺ hepatocytes (PBS group). I, immunostaining for Tunel and tdTomato on liver sections from mice treated with PBS or DT. White scale bar represents 100 μm. J, immunostaining for E-cad and tdTomato on kidney sections of Cyp2e1-DreER;Rosa26-rox-stop-rox-tdTomato (R26-R-tdT) mice and Cyp2e1-DreER;R26-LR-tdT-DTR. Both the mice received tamoxifen. White scale bar represents 100 μm. K, a cartoon figure showing Cyp2e1-DreER targeted hepatocytes specifically by using R26-LR-tdT-DTR mice and AAV8-TBG-Cre injection, while Cyp2e1-DreER targeted hepatocytes and kidney epithelial cells by using R26-R-tdT. Each image is representative of five individual biological samples. DTR, diphtheria toxin receptor; DT, diphtheria toxin.

Figure 6

DTR-mediated specific ablation of Mfsd2a⁺peri-portal hepatocytes.A, a cartoon figure showing the mouse’s mating strategy. B, a schematic figure showing the genetic recombination of the R26-LR-tdT-DTR allele by Mfsd2a-CreER and AAV8-TBG-Dre. C, a schematic figure showing the experimental design. D, whole-mount fluorescent view of livers collected from mice treated with PBS or DT. Yellow scale bar represents 2000 μm. E and F, immunostaining for tdTomato, HNF4a (E), or DTR (F) on liver sections of mice treated with PBS. White scale bar represents100 μm. G, immunostaining for E-cad and tdTomato on liver sections from mice treated with PBS or DT. White scale bar represents 100 μm. H, quantification of the percentage of tdTomato⁺ hepatocytes (DT group) in comparison with tdTomato⁺ hepatocytes (PBS group). n = 5. I, immunostaining for Tunnel and tdTomato on liver sections of mice treated with PBS or DT. White scale bar represents 100 um. J, immunostaining for PECAM, NeuN, E-cad, and tdTomato on tissue sections collected from Mfsd2a-CreER;Rosa26-loxP-stop-loxP-tdTomato (R26-L-tdT) mice and Mfsd2a-CreER;R26-LR-tdT-DTR mice. Both the mice received tamoxifen. White scale bar represents 100 μm. K, a cartoon figure showing Mfsd2a-CreER–targeted hepatocytes specifically by using R26-LR-tdT-DTR mice and AAV8-TBG-Dre injection, whereas Mfsd2a-CreER–targeted hepatocytes, brain neurons, endothelial cells, and intestine epithelial cells by using R26-L-tdT. Each image is a representative of five individual biological samples. DTR, diphtheria toxin receptor.