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Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells
Yang Zhao, … , Y. Eugene Chen, Jifeng Zhang
Yang Zhao, … , Y. Eugene Chen, Jifeng Zhang
Published June 8, 2023
Citation Information: JCI Insight. 2023;8(14):e171661. https://doi.org/10.1172/jci.insight.171661.
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Resource and Technical Advance Angiogenesis Vascular biology

Generating endogenous Myh11-driven Cre mice for sex-independent gene deletion in smooth muscle cells

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Abstract

Specific and efficient smooth muscle cell–targeted (SMC-targeted) gene deletion is typically achieved by pairing SMMHC-CreERT2-Tg mice with mice carrying the loxP-flanked gene. However, the transgene, CreERT2, is not controlled by the endogenous Myh11 gene promoter, and the codon-modified iCreERT2 exhibits significant tamoxifen-independent leakage. Furthermore, because the Cre-bearing bacterial artificial chromosome (BAC) is inserted onto the Y chromosome, the SMMHC-CreERT2-Tg mice strain can only exhibit gene deletions in male mice. Additionally, there is a lack of Myh11-driven constitutive Cre mice when tamoxifen usage is a concern. We used CRISPR/Cas9-mediated homologous recombination between a donor vector carrying the CreNLSP2A or CreERT2–P2A sequence and homologous arm surrounding the translation start site of the Myh11 gene to generate Cre-knockin mice. The P2A sequence enables the simultaneous translation of Cre and endogenous proteins. Using reporter mice, we assessed Cre-mediated recombination efficiency, specificity, tamoxifen-dependent controllability, and functionality in both sexes. Both constitutive (Myh11-CreNLSP2A) and inducible (Myh11-CreERT2–P2A) Cre mice demonstrated efficient, SMC-specific, sex-independent Cre recombinase activity without confounding endogenous gene expression. Combined with recently generated BAC transgenic Myh11-CreERT2-RAD mice and the Itga8-CreERT2 mouse models, our models will help expand the research toolbox, facilitating unbiased and comprehensive research in SMCs and SMC-dependent cardiovascular diseases.

Authors

Yang Zhao, Guizhen Zhao, Ziyi Chang, Tianqing Zhu, Ying Zhao, Haocheng Lu, Chao Xue, Thomas L. Saunders, Yanhong Guo, Lin Chang, Y. Eugene Chen, Jifeng Zhang

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Figure 3

Characterization of tamoxifen-inducible Cre activity in Myh11-CreERT2–P2A KI mice.

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Characterization of tamoxifen-inducible Cre activity in Myh11-CreERT2–P2...
Myh11-CreERT2–P2A KI mice were crossbred with mT/mG or LacZ reporter mice. Mice were i.p. administered 50 mg/kg/day tamoxifen/corn oil for 5 consecutive days, followed by1 week of rest, X-gal staining, frozen sectioning, or protein isolation from different tissues. (A) tdTomato and EGFP signal in frozen sections of aorta, bladder, jejunum, heart, and lower limb isolated from Myh11-CreERT2–P2A+/– or Myh11-CreERT2–P2A–/– mice crossbred with mT/mG reporter mice. Scale bars: 50 μm (aorta, jejunum, heart, skeletal muscle); 100 μm (bladder). L, aortic lumen. (B) Western blot analysis of CreERT2 or EGFP and β-actin in the aorta, bladder, and jejunum, followed by quantification (n = 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (C) X-gal (β-galactosidase) staining to visualize Cre recombinase activity in the aorta, bladder, jejunum, heart, and eWAT from Myh11-CreERT2–P2A+/– or Myh11-CreERT2–P2A–/– mice injected with corn oil or tamoxifen. Data are presented as mean ± SEM. One-way ANOVA for CreERT2 and 2-way ANOVA for EGFP quantification in B, followed by the Tukey test.

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