Tamoxifen-independent recombination of reporter genes limits lineage tracing and mosaic analysis using CreERT2 lines

Abstract

The CreER^T2/loxP system is widely used to induce conditional gene deletion in mice. One of the main advantages of the system is that Cre-mediated recombination can be controlled in time through Tamoxifen administration. This has allowed researchers to study the function of embryonic lethal genes at later developmental timepoints. In addition, CreER^T2 mouse lines are commonly used in combination with reporter genes for lineage tracing and mosaic analysis. In order for these experiments to be reliable, it is crucial that the cell labeling approach only marks the desired cell population and their progeny, as unfaithful expression of reporter genes in other cell types or even unintended labeling of the correct cell population at an undesired time point could lead to wrong conclusions. Here we report that all CreER^T2 mouse lines that we have studied exhibit a certain degree of Tamoxifen-independent, basal, Cre activity. Using Ai14 and Ai3, two commonly used fluorescent reporter genes, we show that those basal Cre activity levels are sufficient to label a significant amount of cells in a variety of tissues during embryogenesis, postnatal development and adulthood. This unintended labelling of cells imposes a serious problem for lineage tracing and mosaic analysis experiments. Importantly, however, we find that reporter constructs differ greatly in their susceptibility to basal CreER^T2 activity. While Ai14 and Ai3 easily recombine under basal CreER^T2 activity levels, mTmG and R26R-EYFP rarely become activated under these conditions and are therefore better suited for cell tracking experiments.

Keywords: Cre/loxP system; CreERT2; Lineage tracing; Mosaic analysis; Reporter-gene; Tamoxifen-independent recombination.

Fig. 1

Principle of the CreER^T2/loxP system and schematic representation of commonly used fluorescent reporter constructs. a In the absence of Tamoxifen, CreER^T2 cannot efficiently enter the nucleus and thus will not recombine target genes. Tamoxifen administration causes CreER^T2 translocation to the nucleus and results in target gene recombination. In the case of the depicted reporter, excision of the loxP flanked stop codon leads to expression of the downstream-located fluorescent reporter gene. b Schematic representation of R26R-EYFP, mTmG, Ai13 and Ai14 reporter lines. LoxP sequences are depicted by triangles. neo^R: neomycin resistance, stop: stop codon, CAG: CAG promoter, WPRE (posttranscriptional regulatory element). The distance in between the loxP sites is indicated for each line (in kb)

Fig. 2

Tamoxifen-independent activation of the Ai14 reporter in primary brain EC isolated from Cdh5(PAC)CreER^T2 mice. a Confocal images of primary brain endothelial cells. Left panel: clusters of EC isolated from Tamoxifen-induced Cdh5(PAC)Cre-ER^T2/Ai14 animals exhibiting a high percentage of tdTomato expressing cells. Middle panel: EC clusters from Tamoxifen-induced mice carrying only the Ai14, but not the Cdh5(PAC)-CreERT2 construct do not show tdTomato expression. Right panel: endothelial clusters from animals that were not Tamoxifen induced but carry both Cdh5(PAC)-CreERT2 and Ai14 show several tdTomato positive cells and reveal a low level of basal Cre activity. Scale bars: 75 μm. b Quantification of the percentage of tdTomato positive cells in cultures of non-Tamoxifen-induced Cdh5(PAC)Cre-ERT2/Ai14 animals one and three days after isolation. ***P ≤ 0.001

Fig. 3

Several CreER^T2 lines are basally active and lead to reporter gene activation in vivo in the absence of Tamoxifen. a Confocal images of the ear skin vasculature of mice carrying the Ai14 reporter alone or in combination with one of the following CreER^T2 lines: Prox1 (expressed in lymphatic endothelial cells, LEC), Pdgfb (expressed in LEC and blood endothelial cells, BEC) or Cdh5 (expressed in LEC and BEC). Widespread tdTomato expression, a consequence of Tamoxifen independent recombination of the Ai14 reporter, is observed in all CreER^T2 lines, but it does not occur in the absence of a CreER^T2 driver in mice that only carry the Ai14 reporter. BEC and LEC are visualized with a CD31 antibody (red) and LEC are stained with a Lyve1 antibody (blue). tdTomato expression from the Ai14 reporter is shown in green. Scale bars indicate 500 μm. b Higher resolution images from the pictures in a). Scale bars indicate 200 μm

Fig. 4

Fluorescent reporters differ in their susceptibility to basal CreER^T2 activity. a Representative confocal images of whole mount retinas from animals carrying Cdh5(PAC)-CreER^T2 and either Ai14, Ai3, mTmG, or the R26R-EYFP reporter. Endothelial cells are labeled with IB4 (isolectin B4), in red, and fluorescent proteins expressed by the individual reporters are depicted in green. Left column panels: Both the Ai14 and the Ai3 reporters appear to have a low recombination threshold, as basal (non-Tamoxifen induced) Cdh5(PAC)-CreER^T2 levels are sufficient to induce expression of the fluorescent reporters in a significant amount of cells. In contrast, only few or no fluorescent cells were observed in mTmG or R26R-EYFP retinas, indicating that those reporters recombine inefficiently under basal CreER^T2 leakage-levels. Scale bars indicate 200 μm. Right column panels: detail of the areas marked with white squares in the left panels. Scale bars indicate 50 μm. b Quantification of fluorescent cells found in the retinas of the different reporter mice (Ai14: n = 5, Ai3: n = 7, mTmG: n = 6, R26R-EYFP: n = 5). One way ANOVA was performed for statistical analysis. c Extreme example of the degree of reporter activation that can be observed under basal, non-Tamoxifen-induced, conditions using Cdh5(PAC)-CreER^T2 in combination with Ai14. The vasculature is labeled by IB4 in red, and the Ai14 reporter signal is shown in green. Scale bar indicates 500 μm

Fig. 5

Activation of fluorescent reporters under basal CreER^T2 activity levels in the brain. a Left column panels: representative images of brains sections from Cdh5(PAC)-CreER^T2 mice with either the Ai14, Ai3, mTmG, or R26R-EYFP reporter. Positive endothelial cells can be seen in all brains to varying degrees. Scale bars indicate 100 μm. Right column panels: detailed images of the areas marked with squares in the left panels. Scale bars indicate 50 μm. b Quantification of fluorescent cells in brain sections of the different reporter mice (Ai14: n = 5, Ai3: n = 5, mTmG: n = 5, R26R-EYFP: n = 7). One way ANOVA was performed for statistical analysis. c PCR analysis of Cre-mediated recombination in the Ai14 and R26R-EYFP reporter. Two sets of PCR primers (P1 and P2) were designed to flank the loxP sites (indicated by black arrowheads) with P1 priming upstream of the first loxP site and P2 within the fluorescent gene (indicated in green, for Ai14 and R26R-EYFP respectively). PCRs conditions were optimized to amplify the genomic DNA region that remained after recombination had occurred: 291 bp (Ai14) and 775 bp (R26R-EYFP). After 38 cycles, abundant PCR product was obtained for both Ai14 and R26R-EYFP after Tamoxifen induction, while in the absence of Tamoxifen only PCR product from the Ai14 reporter was detected. After 41 cycles, increased amount of PCR product was obtained for both Ai14 and R26R-EYFP after Tamoxifen induction, and in the non-Tamoxifen-treated Ai14 sample, whereas only a very faint band appeared in the non-Tamoxifen-treated R26R-EYFP sample. In the absence of CreER^T2, no product is detected in any PCR

Fig. 6

Transient CreER^T2 expression and leakage in an ancestor cell may hamper lineage tracing in daughter populations. a Fluorescent reporter expression in brain endothelial cells and microglia as a result of basal CreER^T2 leakage in mice carrying Cdh5(PAC)-CreER^T2 and the Ai3 reporter. Endothelial cells are shown in blue (CD31) and microglia are depicted in red (Iba1). Note that Ai3 (shown in green), is labeling a subset of endothelial cells and microglia. Lower panels represent microglial staining and Ai3 signal, respectively. Scale bars indicate 20 μm. b Fluorescent reporter expression in retinas of mice carrying Cdh5(PAC)-CreER^T2 and the Ai14 reporter. Upper row: reporter expression in microglia due to basal CreER^T2 leakage. Lower row: representative image of the retina of a Tamoxifen-induced mouse, note the absence of positive microglia in this retina. Vascular outlines are shown in red (isolectin B4 or CD31) and Ai14 reporter expression is shown in green. Scale bars indicate 25 μm

Fig. 7

Properties of the tested reporter constructs and recommendations for preferred usage. Diagram depicting brightness differences of the fluorescent proteins in the analyzed reporters as well as their recombination threshold, based on basal CreER^T2 leakage in non-tamoxifen induced animals. Ai14 is the brightest reporter tested and thus well suited for live cell-imaging approaches. The fact that it easily recombines makes it suitable for experiments in which one aims for a maximum of cells to be labeled. The R26R-EYFP reporter is difficult to image unless amplified by immunohistochemistry and therefore less suited for live cell imaging. However, the R26R-EYFP and the mTmG reporter are less susceptible to basal CreER^T2 leakage