The Cre–LoxP system provides a widely used method for studying gene requirements in the mouse as the main mammalian genetic model organism. To define the molecular and cellular mechanisms that underlie cardiovascular development, function and disease, various mouse strains have been engineered that allow Cre–LoxP-mediated gene targeting within specific cell types of the cardiovascular system. Despite the usefulness of this system, evidence is accumulating that Cre activity can have toxic effects in cells, which are independent of its ability to recombine pairs of engineered LoxP sites in target genes. Here, we have gathered published evidence for Cre toxicity in cells and tissues relevant to cardiovascular biology and provide an overview of mechanisms proposed to underlie Cre toxicity. Based on this knowledge, we propose that each study utilizing the Cre–LoxP system to investigate gene function in the cardiovascular system should incorporate appropriate controls to account for Cre toxicity.
This is a preview of subscription content, access via your institution
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Orban, P. C., Chui, D. & Marth, J. D. Tissue- and site-specific DNA recombination in transgenic mice. Proc. Natl Acad. Sci. USA 89, 6861–6865 (1992).
Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).
Agah, R. et al. Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J. Clin. Invest. 100, 169–179 (1997).
Schmidt, A. et al. lacZ transgenic mice to monitor gene expression in embryo and adult. Brain Res. Brain Res. Protoc. 3, 54–60 (1998).
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).
Li, S. et al. Overview of the reporter genes and reporter mouse models. Animal Model. Exp. Med. 1, 29–35 (2018).
Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).
Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).
Chen, C. M., Krohn, J., Bhattacharya, S. & Davies, B. A comparison of exogenous promoter activity at the ROSA26 locus using a PhiiC31 integrase mediated cassette exchange approach in mouse ES cells. PLoS ONE 6, e23376 (2011).
Kawamoto, S. et al. A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination. FEBS Lett. 470, 263–268 (2000).
Sakai, K. & Miyazaki, J. A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem. Biophys. Res. Commun. 237, 318–324 (1997).
Heffner, C. S. et al. Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource. Nat. Commun. 3, 1218 (2012).
Alva, J. A. et al. VE-Cadherin-Cre-recombinase transgenic mouse: a tool for lineage analysis and gene deletion in endothelial cells. Dev. Dyn. 235, 759–767 (2006).
Hayashi, S. & McMahon, A. P. Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev. Biol. 244, 305–318 (2002).
Feil, R. et al. Ligand-activated site-specific recombination in mice. Proc. Natl Acad. Sci. USA 93, 10887–10890 (1996).
Metzger, D., Clifford, J., Chiba, H. & Chambon, P. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc. Natl Acad. Sci. USA 92, 6991–6995 (1995).
Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752–757 (1997).
Littlewood, T. D., Hancock, D. C., Danielian, P. S., Parker, M. G. & Evan, G. I. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 23, 1686–1690 (1995).
Zhang, Y. et al. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res. 24, 543–548 (1996).
Valny, M., Honsa, P., Kirdajova, D., Kamenik, Z. & Anderova, M. Tamoxifen in the mouse brain: implications for fate-mapping studies using the tamoxifen-inducible Cre–loxP system. Front. Cell. Neurosci.10, 243 (2016).
Zhong, Q. et al. Boronic prodrug of 4-hydroxytamoxifen is more efficacious than tamoxifen with enhanced bioavailability independent of CYP2D6 status. BMC Cancer 15, 625 (2015).
Thanos, A. et al. Evidence for baseline retinal pigment epithelium pathology in the Trp1-Cre mouse. Am. J. Pathol. 180, 1917–1927 (2012).
Lam, P. T. et al. Considerations for the use of Cre recombinase for conditional gene deletion in the mouse lens. Hum. Genomics 13, 10 (2019).
Amin, S. R. et al. Viral vector-mediated Cre recombinase expression in substantia nigra induces lesions of the nigrostriatal pathway associated with perturbations of dopamine-related behaviors and hallmarks of programmed cell death. J. Neurochem. 150, 330–340 (2019).
Forni, P. E. et al. High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly. J. Neurosci. 26, 9593–9602 (2006).
Jeannotte, L. et al. Unsuspected effects of a lung-specific Cre deleter mouse line. Genesis 49, 152–159 (2011).
Balkawade, R. S. et al. Podocyte-specific expression of Cre recombinase promotes glomerular basement membrane thickening. Am. J. Physiol. Renal Physiol. 316, F1026–F1040 (2019).
Bohin, N., Carlson, E. A. & Samuelson, L. C. Genome toxicity and impaired stem cell function after conditional activation of CreERT2 in the intestine. Stem Cell Reports 11, 1337–1346 (2018).
Huh, W. J., Mysorekar, I. U. & Mills, J. C. Inducible activation of Cre recombinase in adult mice causes gastric epithelial atrophy, metaplasia, and regenerative changes in the absence of ‘floxed’ alleles. Am. J. Physiol. Gastrointest. Liver Physiol. 299, G368–G380 (2010).
Li, Y., Choi, P. S., Casey, S. C. & Felsher, D. W. Activation of Cre recombinase alone can induce complete tumor regression. PLoS ONE 9, e107589 (2014).
Janbandhu, V. C., Moik, D. & Fassler, R. Cre recombinase induces DNA damage and tetraploidy in the absence of loxP sites. Cell Cycle 13, 462–470 (2014).
Ng, W. A., Grupp, I. L., Subramaniam, A. & Robbins, J. Cardiac myosin heavy chain mRNA expression and myocardial function in the mouse heart. Circ. Res. 68, 1742–1750 (1991).
Sohal, D. S. et al. Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein. Circ. Res. 89, 20–25 (2001).
Pugach, E. K., Richmond, P. A., Azofeifa, J. G., Dowell, R. D. & Leinwand, L. A. Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic. J. Mol. Cell. Cardiol. 86, 54–61 (2015).
Garbern, J. et al. Analysis of Cre-mediated genetic deletion of Gdf11 in cardiomyocytes of young mice. Am. J. Physiol. Heart. Circ. Physiol. 317, H201–H212 (2019).
McLean, B. A. et al. PI3Kα is essential for the recovery from Cre/tamoxifen cardiotoxicity and in myocardial insulin signalling but is not required for normal myocardial contractility in the adult heart. Cardiovasc. Res. 105, 292–303 (2015).
Lexow, J., Poggioli, T., Sarathchandra, P., Santini, M. P. & Rosenthal, N. Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver. Dis. Model Mech. 6, 1470–1476 (2013).
Bersell, K. et al. Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death. Dis. Model Mech. 6, 1459–1469 (2013).
Kisanuki, Y. Y. et al. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230, 230–242 (2001).
Theis, M. et al. Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene. Genesis 29, 1–13 (2001).
Sorensen, I., Adams, R. H. & Gossler, A. DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries. Blood 113, 5680–5688 (2009).
Okabe, K. et al. Neurons limit angiogenesis by titrating VEGF in retina. Cell 159, 584–596 (2014).
Claxton, S. et al. Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46, 74–80 (2008).
Payne, S., Val, S. D. & Neal, A. Endothelial-specific Cre mouse models. Arterioscler. Thromb. Vasc. Biol. 38, 2550–2561 (2018).
Brash, J., Bolton, R., Rashbrook, V., Denti, L. & Ruhrberg, C. Tamoxifen-activated CreERT impairs retinal angiogenesis independently of gene deletion. Circ. Res. https://doi.org/10.1161/CIRCRESAHA.120.317025 (2020).
Kanki, Y. et al. Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis. Cell Rep. 38, 110332 (2022).
Ruhrberg, C. & Bautch, V. L. Neurovascular development and links to disease. Cell. Mol. Life Sci. 70, 1675–1684 (2013).
Pitulescu, M. E., Schmidt, I., Benedito, R. & Adams, R. H. Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat. Protoc. 5, 1518–1534 (2010).
Powner, M. B. et al. Visualization of gene expression in whole mouse retina by in situ hybridization. Nat. Protoc. 7, 1086–1096 (2012).
Toullec, A. et al. HIF-1α deletion in the endothelium, but not in the epithelium, protects from radiation-induced enteritis. Cell Mol. Gastroenterol. Hepatol. 5, 15–30 (2018).
Mohamed, R. et al. Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction. Biochim. Biophys. Acta Mol. Basis Dis. 1864, 746–757 (2018).
Naldini, A. & Carraro, F. Role of inflammatory mediators in angiogenesis. Curr. Drug Targets Inflamm. Allergy 4, 3–8 (2005).
Limbourg, A. et al. MAP-kinase activated protein kinase 2 links endothelial activation and monocyte/macrophage recruitment in arteriogenesis. PLoS ONE 10, e0138542 (2015).
Ruparelia, N., Chai, J. T., Fisher, E. A. & Choudhury, R. P. Inflammatory processes in cardiovascular disease: a route to targeted therapies. Nat. Rev. Cardiol. 14, 133–144 (2017).
Higashi, A. Y. et al. Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2. J. Immunol. 182, 5633–5640 (2009).
Kurachi, M., Ngiow, S. F., Kurachi, J., Chen, Z. & Wherry, E. J. Hidden caveat of inducible Cre recombinase. Immunity 51, 591–592 (2019).
Zeitrag, J., Alterauge, D., Dahlstrom, F. & Baumjohann, D. Gene dose matters: considerations for the use of inducible CD4–CreERT2 mouse lines. Eur. J. Immunol. 50, 603–605 (2020).
Popov, D. Endothelial cell dysfunction in hyperglycemia: phenotypic change, intracellular signaling modification, ultrastructural alteration, and potential clinical outcomes. Int. J. Diabetes Mellit. 2, 189–195 (2010).
Sweet, I. R. et al. Endothelial inflammation induced by excess glucose is associated with cytosolic glucose 6-phosphate but not increased mitochondrial respiration. Diabetologia 52, 921–931 (2009).
Hempel, A. et al. High glucose concentrations increase endothelial cell permeability via activation of protein kinase C alpha. Circ. Res. 81, 363–371 (1997).
Karbach, S. et al. Hyperglycemia and oxidative stress in cultured endothelial cells–a comparison of primary endothelial cells with an immortalized endothelial cell line. J Diabetes Complications 26, 155–162 (2012).
Duvillie, B. et al. Phenotypic alterations in insulin-deficient mutant mice. Proc. Natl Acad. Sci. USA 94, 5137–5140 (1997).
Lee, J. Y. et al. RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function. J. Biol. Chem. 281, 2649–2653 (2006).
Pomplun, D., Florian, S., Schulz, T., Pfeiffer, A. F. & Ristow, M. Alterations of pancreatic beta-cell mass and islet number due to Ins2-controlled expression of Cre recombinase: RIP-Cre revisited; part 2. Horm. Metab. Res. 39, 336–340 (2007).
Loonstra, A. et al. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc. Natl Acad. Sci. USA 98, 9209–9214 (2001).
Silver, D. P. & Livingston, D. M. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol. Cell 8, 233–243 (2001).
Thyagarajan, B., Guimaraes, M. J., Groth, A. C. & Calos, M. P. Mammalian genomes contain active recombinase recognition sites. Gene 244, 47–54 (2000).
Semprini, S. et al. Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques. Nucleic Acids Res. 35, 1402–1410 (2007).
Norbury, C. J. & Zhivotovsky, B. DNA damage-induced apoptosis. Oncogene 23, 2797–2808 (2004).
Xiao, Y. et al. Cre-mediated stress affects sirtuin expression levels, peroxisome biogenesis and metabolism, antioxidant and proinflammatory signaling pathways. PLoS ONE 7, e41097 (2012).
Zhu, J., Nguyen, M. T., Nakamura, E., Yang, J. & Mackem, S. Cre-mediated recombination can induce apoptosis in vivo by activating the p53 DNA damage-induced pathway. Genesis 50, 102–111 (2012).
Wang, X., Lauth, A., Wan, T. C., Lough, J. W. & Auchampach, J. A. Myh6-driven Cre recombinase activates the DNA damage response and the cell cycle in the myocardium in the absence of loxP sites. Dis. Model Mech. https://doi.org/10.1242/dmm.046375 (2020).
Rehmani, T., Salih, M. & Tuana, B. S. Cardiac-specific cre induces age-dependent dilated cardiomyopathy (DCM) in mice. Molecules https://doi.org/10.3390/molecules24061189 (2019).
Pfeifer, A., Brandon, E. P., Kootstra, N., Gage, F. H. & Verma, I. M. Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo. Proc. Natl Acad. Sci. USA 98, 11450–11455 (2001).
Baba, Y., Nakano, M., Yamada, Y., Saito, I. & Kanegae, Y. Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells. Microbiol. Immunol. 49, 559–570 (2005).
Naiche, L. A. & Papaioannou, V. E. Cre activity causes widespread apoptosis and lethal anemia during embryonic development. Genesis 45, 768–775 (2007).
Gangoda, L. et al. Cre transgene results in global attenuation of the cAMP/PKA pathway. Cell Death Dis. 3, e365 (2012).
Sassone-Corsi, P. The cyclic AMP pathway. Cold Spring Harb. Perspect. Biol. https://doi.org/10.1101/cshperspect.a011148 (2012).
Pepin, G. et al. Cre-dependent DNA recombination activates a STING-dependent innate immune response. Nucleic Acids Res. 44, 5356–5364 (2016).
Gerhart-Hines, Z. et al. The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD+. Mol Cell 44, 851–863 (2011).
Merksamer, P. I. et al. The sirtuins, oxidative stress and aging: an emerging link. Aging 5, 144–150 (2013).
Roth, M. & Chen, W. Y. Sorting out functions of sirtuins in cancer. Oncogene 33, 1609–1620 (2014).
Potente, M. et al. SIRT1 controls endothelial angiogenic functions during vascular growth. Genes Dev. 21, 2644–2658 (2007).
Kitada, M., Ogura, Y. & Koya, D. The protective role of Sirt1 in vascular tissue: its relationship to vascular aging and atherosclerosis. Aging 8, 2290–2307 (2016).
Goodwin, L. O. et al. Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome Res. 29, 494–505 (2019).
Cain-Hom, C. et al. Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification. Nucleic Acids Res. 45, e62 (2017).
Lewis, A. E., Vasudevan, H. N., O’Neill, A. K., Soriano, P. & Bush, J. O. The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling. Dev. Biol. 379, 229–234 (2013).
Harkins, S. & Whitton, J. L. Chromosomal mapping of the αMHC–MerCreMer transgene in mice reveals a large genomic deletion. Transgenic Res. 25, 639–648 (2016).
Declercq, J. et al. Metabolic and behavioural phenotypes in nestin-Cre mice are caused by hypothalamic expression of human growth hormone. PLoS ONE 10, e0135502 (2015).
Fernandez-Chacon, M. et al. iSuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications. Nat. Commun. 10, 2262 (2019).
Ved, N., Curran, A., Ashcroft, F. M. & Sparrow, D. B. Tamoxifen administration in pregnant mice can be deleterious to both mother and embryo. Lab. Anim. 53, 630–633 (2019).
Wyatt, K. D., Sakamoto, K. & Watford, W. T. Tamoxifen administration induces histopathologic changes within the lungs of Cre-recombinase-negative mice: a case report. Lab. Anim. https://doi.org/10.1177/00236772211042968 (2021).
Keeley, T. M., Horita, N. & Samuelson, L. C. Tamoxifen-induced gastric injury: effects of dose and method of administration. Cell Mol. Gastroenterol. Hepatol. 8, 365–367 (2019).
Patel, S. H. et al. Low-dose tamoxifen treatment in juvenile males has long-term adverse effects on the reproductive system: implications for inducible transgenics. Sci Rep. 7, 8991 (2017).
Sun, M. R., Steward, A. C., Sweet, E. A., Martin, A. A. & Lipinski, R. J. Developmental malformations resulting from high-dose maternal tamoxifen exposure in the mouse. PLoS ONE 16, e0256299 (2021).
Ahn, S. H. et al. Tamoxifen suppresses pancreatic beta-cell proliferation in mice. PLoS ONE 14, e0214829 (2019).
Alsina-Sanchis, E. et al. Intraperitoneal oil application causes local inflammation with depletion of resident peritoneal macrophages. Mol. Cancer Res. 19, 288–300 (2021).
Brash, J. T., Denti, L., Ruhrberg, C. & Bucher, F. VEGF188 promotes corneal reinnervation after injury. JCI Insight https://doi.org/10.1172/jci.insight.130979 (2019).
Fantin, A. et al. NRP1 acts cell autonomously in endothelium to promote tip cell function during sprouting angiogenesis. Blood 121, 2352–2362 (2013).
Jahn, H. M. et al. Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia. Sci Rep. 8, 5913 (2018).
Carlos-Reyes, A., Muniz-Lino, M. A., Romero-Garcia, S., Lopez-Camarillo, C. & Hernandez-de la Cruz, O. N. Biological adaptations of tumor cells to radiation therapy. Front. Oncol. 11, 718636 (2021).
Wilm, T. P. et al. Restricted differentiative capacity of Wt1-expressing peritoneal mesothelium in postnatal and adult mice. Sci Rep. 11, 15940 (2021).
Zorn, A. M. & Wells, J. M. Vertebrate endoderm development and organ formation. Annu. Rev. Cell Dev. Biol. 25, 221–251 (2009).
Stearns, V. et al. Active tamoxifen metabolite plasma concentrations after coadministration of tamoxifen and the selective serotonin reuptake inhibitor paroxetine. J. Natl Cancer Inst. 95, 1758–1764 (2003).
Fan, Q., Mao, H., Xie, L. & Pi, X. Prolyl hydroxylase domain-2 protein regulates lipopolysaccharide-induced vascular inflammation. Am. J. Pathol. 189, 200–213 (2019).
Ding, B. S. et al. Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell 147, 539–553 (2011).
Hameyer, D. et al. Toxicity of ligand-dependent Cre recombinases and generation of a conditional Cre deleter mouse allowing mosaic recombination in peripheral tissues. Physiol. Genomics 31, 32–41 (2007).
Kim, J. H. et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE 6, e18556 (2011).
Jakobsson, L. et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat. Cell Biol. 12, 943–953 (2010).
Luo, W. et al. Arterialization requires the timely suppression of cell growth. Nature 589, 437–441 (2021).
Meuwissen, R., Linn, S. C., van der Valk, M., Mooi, W. J. & Berns, A. Mouse model for lung tumorigenesis through Cre/lox controlled sporadic activation of the K-Ras oncogene. Oncogene 20, 6551–6558 (2001).
Han, X. et al. A suite of new Dre recombinase drivers markedly expands the ability to perform intersectional genetic targeting. Cell Stem Cell https://doi.org/0.1016/j.stem.2021.01.007 (2021).
Tycko, J. et al. Mitigation of off-target toxicity in CRISPR–Cas9 screens for essential noncoding elements. Nat. Commun. 10, 4063 (2019).
V.S.R. and J.T.B. were supported by the British Heart Foundation (PG/19/37/3439 and FS/18/65/34186), and C.R. by Wellcome (205099/Z/16/Z).
The authors declare no competing interests.
Peer review information
Nature Cardiovascular Research thanks Bin Zhou, Rui Benedito and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Rashbrook, V.S., Brash, J.T. & Ruhrberg, C. Cre toxicity in mouse models of cardiovascular physiology and disease. Nat Cardiovasc Res 1, 806–816 (2022). https://doi.org/10.1038/s44161-022-00125-6
This article is cited by
Nature Cardiovascular Research (2022)