Cre recombinase induces DNA damage and tetraploidy in the absence of<i>LoxP</i>sites

Janbandhu, Vaibhao; Moik, Daniel; Fässler, Reinhard

doi:10.4161/cc.27271

Cited by 91 publications

(66 citation statements)

References 65 publications

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“…The cre-loxP system is widely used in mammalian systems to cause targeted deletion of DNA segments from the genome. Although this system is potentially very useful, experiments using the cre-loxP system can be complicated by the off target effects of cre recombinase [29, 44, 45]. …”

Section: Discussionmentioning

confidence: 99%

Estrogen receptor alpha promotes lupus in (NZB×NZW)F1 mice in a B cell intrinsic manner

Tabor

Gould

2017

Clinical Immunology

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Lupus is a systemic autoimmune disease characterized by the production of autoreactive antibodies against nuclear antigens. Women are disproportionately affected by lupus, and this sex bias is thought to be due, in large part, to the ability of estrogens to promote lupus pathogenesis. Previously, we have shown that global deletion of estrogen receptor alpha (ERα) significantly attenuated loss of tolerance, immune cell activation, autoantibody production, and the development of lupus nephritis. Here we show that targeted deletion of ERα specifically in B cells retards production of pathogenic autoantibodies and the development of nephritis in lupus-prone (NZB x NZW)F1 mice. Furthermore, we observed that ERα deletion in B cells was associated with decreased B cell activation in young, pre-autoimmune (NZB x NZW)F1 females. Altogether, these data suggest that ERα acts in a B cell-intrinsic manner to control B cell activation, autoantibody production, and lupus nephritis.

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Section: Discussionmentioning

confidence: 99%

Estrogen receptor alpha promotes lupus in (NZB×NZW)F1 mice in a B cell intrinsic manner

Tabor

Gould

2017

Clinical Immunology

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“…However, given prolonged, high Cre expression, Cre’s role as a DNA recombinase with promiscuous activity, and the presence of degenerate loxP sites in the mouse genome (Figure 4), we suggest this as a potential mechanism of cardiotoxicity in conjunction with an inflammatory response activated by long-term exposure to a non-endogenous DNA recombinase. Further, in a skin model of Cre toxicity, DNA damage response was shown to be dependent on Cre recombinase activity as an endonuclease-deficient isoform of Cre did not activate this pathway [9]. …”

Section: Discussionmentioning

confidence: 99%

“…In non-cardiac tissue cell-types, Cre expression has been shown to induce DNA damage and apoptosis in the absence of bona fide loxP sites [9,10]. Toxic effects associated with Cre expression have been observed in gastrointestinal cells, neurons, and spermatids [10–12].…”

Section: Introductionmentioning

confidence: 99%

Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic

Pugach

Richmond

Azofeifa

et al. 2015

Journal of Molecular and Cellular Cardiology

101

105

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Studying the importance of genetic factors in a desired cell type or tissue necessitates the use of precise genetic tools. With the introduction of bacteriophage Cre recombinase/loxP mediated DNA editing and promoter-specific Cre expression, it is feasible to generate conditional knockout mice in which particular genes are disrupted in a cell type-specific manner in vivo. In cardiac myocytes, this is often achieved through α-myosin heavy chain promoter (αMyHC)-driven Cre expression in conjunction with a loxP-site flanked gene of interest. Recent studies in other cell types demonstrate toxicity of Cre expression through induction of DNA damage. However, it is unclear to what extent the traditionally used αMyHC-Cre line [1] may exhibit cardiotoxicity. Further, the genotype of αMyHC-Cre+/− is not often included as a control group in cardiac myocyte-specific knockout studies. Here we present evidence that these αMyHC-Cre+/− mice show molecular signs of cardiac toxicity by 3 months of age and exhibit decreased cardiac function by 6 months of age compared to wild-type littermates. Hearts from αMyHC-Cre+/− mice also display evidence of fibrosis, inflammation, and DNA damage. Interestingly, some of the early functional changes observed in αMyHC-Cre+/− mice are sexually dimorphic. Given the high level of Cre recombinase expression resulting from expression from the αMyHC promoter, we asked if degenerate loxP-like sites naturally exist in the mouse genome and if so, whether they are affected by Cre in the absence of canonical loxP-sites. Using a novel bioinformatics search tool, we identified 619 loxP-like sites with 4 or less mismatches to the canonical loxP-site. 227 sites overlapped with annotated genes and 55 of these genes were expressed in cardiac muscle. Expression of ~26% of the 27 genes tested was disrupted in αMyHC-Cre+/− mice indicating potential targeting by Cre. Taken together, these results highlight both the importance of using αMyHC-Cre mice as controls in conditional knockout studies as well as the need for a less cardiotoxic Cre driver for the field.

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“…16–18 This cre toxicity is the result of cre-mediated DNA cleavage at pseudo-loxP sites, which are found relatively abundantly throughout the murine genome. 24 The increased T cell apoptosis in Lck-cre (NZB × NZW)F1 mice lead to a decrease in the relative abundance of certain T cell subsets.…”

Section: Discussionmentioning

confidence: 99%

An Lck–cre transgene accelerates autoantibody production and lupus development in (NZB × NZW)F1 mice

Nelson

Gould

2015

Lupus

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Lupus is an autoimmune disease characterized by the development of antinuclear autoantibodies and immune complex-mediated tissue damage. T cells in lupus patients appear to undergo apoptosis at an increased rate, and this enhanced T cell apoptosis has been postulated to contribute to lupus pathogenesis by increasing autoantigen load. However, there is no direct evidence to support this hypothesis. In this study, we show that an Lck-cre transgene, which increases T cell apoptosis as a result of T cell specific expression of cre recombinase, accelerates the development of autoantibodies and nephritis in lupus-prone (NZB×NZW)F1 mice. Although the enhanced T cell apoptosis in Lck-cre transgenic mice resulted in an overall decrease in the relative abundance of splenic CD4+ and CD8+ T cells, the proportion of activated CD4+ T cells was increased and no significant change was observed in the relative abundance of suppressive T cells. We postulate that the Lck-cre transgene promoted lupus by enhancing T cells apoptosis, which, in conjunction with the impaired clearance of apoptotic cells in lupus-prone mice, increased the nuclear antigen load and accelerated the development of anti-nuclear autoantibodies. Furthermore, our results also underscore the importance of including cre-only controls in studies using the cre-lox system.

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Cre recombinase induces DNA damage and tetraploidy in the absence ofLoxPsites

Cited by 91 publications

References 65 publications

Estrogen receptor alpha promotes lupus in (NZB×NZW)F1 mice in a B cell intrinsic manner

Estrogen receptor alpha promotes lupus in (NZB×NZW)F1 mice in a B cell intrinsic manner

Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic

An Lck–cre transgene accelerates autoantibody production and lupus development in (NZB × NZW)F1 mice

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