Tamoxifen-independent Cre-activity in SMMHC-CreER T2 mice

Abstract

Background and aims: Recent technological advances have established vascular smooth muscle cells (SMCs) as central players in atherosclerosis. Increasingly complex genetic mouse models have unveiled that 30-70% of cells in experimentally induced atherosclerotic lesions derive from a handful of medial SMCs, and that these can adopt a broad range of plaque cell phenotypes. Most of these models are based on the SMMHC-CreER ^T2 mouse line as Cre-driver. Importantly, Cre-activation can be controlled in time (by administration of tamoxifen, TAM), which is critical to avoid unwanted effects of premature recombination events. The aim of this study was to scrutinize an unexpected observation of TAM-independent Cre-activity in this mouse line.

Methods: Cre-activity was assessed by PCR in tissues from SMMHC-CreER ^T2 mice crossed with mice homozygous for loxP-flanked (floxed) exon 4 of Ccn2 (our gene-of-interest), and Ccn2 protein was measured in aortas by targeted mass spectrometry.

Results: We observed spontaneous near-complete excision of floxed Ccn2 in aortas from adult mice that were not treated with TAM. As a result, Ccn2 protein was significantly reduced in aortas from these mice, but not to the same extent as TAM-treated littermates. Remarkably, most of the excision was completed in 4-week-old mice. Excision was Cre-dependent, as knockout bands were negligible in heart and liver (dominated by non-SMCs) of these mice, and undetectable in the aorta in the absence of Cre.

Conclusion: Our observations warrant caution, and we advocate inclusion of appropriate controls (i.e., TAM-untreated mice) in future studies.

Keywords: Atherosclerosis; Cre-loxP; Mouse; Smooth muscle cell.

Fig. 1

a. In smooth muscle cells, CreER^T2 expression is driven by the SMMHC promoter and CreER^T2 is sequestered from the nucleus in the absence of tamoxifen (TAM). Upon TAM-treatment, CreER^T2 binds TAM and translocates to the nucleus. I-III: Three examples of CreER^T2-loxP system use. I: Cre excises (knocks out) a floxed gene-of-interest (GOI), II: Cre excises a STOP cassette leading to transcription of a transgene (e.g., a reporter gene), III: Cre inverts the open reading frame of a transgene resulting in transgene activation. loxP sites are shown as triangles. p = promoter; S = STOP cassette. b.Ccn2 locus genotyping of aortas from TAM-treated and -untreated wildtype (wt) and Ccn2^fl/fl/SMMHC-CreER^T2 males (n = 3 per group, randomly sampled from experiment including n = 6-9 mice per group). Amplicons representing intact (non-excised) flCcn2 (1003 bp), wt Ccn2 (878 bp), and excised flCcn2 (587 bp) are separated by gel electrophoresis and shown on the figure. Marker bands indicated by grey arrowheads are from top: 1500 bp, 1000 bp, 700 bp, 500 bp (consistent throughout gels of Fig. 1). ntc = no template control. c. Targeted mass spectrometry of Ccn2 (normalized to Gapdh) in aortas from the same experiment as in Fig. 1b. Data represent mean ± SEM. ∗ = p ≤ 0.05, ∗ = p ≤ 0.001 (Tukey's multiple comparisons test following one-way ANOVA). The peptides targeted in the assay and their position in Ccn2 and Gapdh, respectively, are shown to the right. d.Ccn2 locus genotyping of aorta, heart, and liver from TAM-untreated Ccn2^fl/fl/SMMHC-CreER^T2 males at the ages indicated. e. Quantitative analysis of Ccn2 locus PCRs (same samples as in Fig. 1d) separated by capillary electrophoresis. The percentages of intact and excised flCcn2 (moles of each amplicon relative to moles of both amplicons), respectively, are shown for aorta, heart and liver. Data are shown as means. f.Ccn2 locus genotyping of tail biopsies from the time of unweaning (3 weeks of age), and tail biopsies and aortas from the time of sacrifice (10 weeks of age) from four mice.