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

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 a-myosin heavy chain promoter (alpha MyHC)-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 alpha MyHC-Cre line [1] may exhibit cardiotoxicity. Further, the genotype of alpha MyHC-Cre(+/-) is not often included as a control group in cardiac myocyte-specific knockout studies. Here we present evidence that these alpha MyHC-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 aMyHC-Cre(+/-) mice also display evidence of fibrosis, inflammation, and DNA damage. Interestingly, some of the early functional changes observed in alpha MyHC-Cre(+/-) mice are sexually dimorphic. Given the high level of Cre recombinase expression resulting from expression from the alpha MyHC 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 similar to 26% of the 27 genes tested was disrupted in alpha MyHC-Cre(+/-) mice indicating potential targeting by Cre. Taken together, these results highlight both the importance of using alpha MyHC-Cre mice as controls in conditional knockout studies as well as the need for a less cardiotoxic Cre driver for the field. (C) 2015 Elsevier Ltd. All rights reserved.