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Myh6 promoter-driven Cre recombinase excises floxed DNA fragments in a subset of male germline cells.

ABSTRACT: Myh6-Cre transgenic mouse line was known to express Cre recombinase only in the heart. Nevertheless, during breeding Myh6-Cre to Rosa26^fstdTom reporter (tdTom) mouse line, we observed that a significant part of their F2 tdTom/+ offspring had tdTom reporter gene universally activated. Our results show that Myh6-Cre transgenic mice have Cre recombinase activity in a subpopulation of the male germline cells, and that Myh6 gene transcripts are enriched in the interstitial Leydig cells and the undifferentiated spermatogonia stem cells. In summary, the current study confirms that the previously known "heart-specific" Myh6 promoter drives Cre expression in the testis.

SUBMITTER: Sheldon C

PROVIDER: S-EPMC9974737 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Myh6 promoter-driven Cre recombinase excises floxed DNA fragments in a subset of male germline cells.

Sheldon Caroline C Kessinger Chase W CW Sun Yan Y Kontaridis Maria I MI Ma Qianyi Q Hammoud Saher Sue SS Gao Zibei Z Zhang Hui H Lin Zhiqiang Z

Journal of molecular and cellular cardiology 20221228

Myh6-Cre transgenic mouse line was known to express Cre recombinase only in the heart. Nevertheless, during breeding Myh6-Cre to Rosa26<sup>fstdTom</sup> reporter (tdTom) mouse line, we observed that a significant part of their F2 tdTom/+ offspring had tdTom reporter gene universally activated. Our results show that Myh6-Cre transgenic mice have Cre recombinase activity in a subpopulation of the male germline cells, and that Myh6 gene transcripts are enriched in the interstitial Leydig cells and ...[more]

PMID: 36584478

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Project description:The Cre-loxP strategy for tissue-specific gene inactivation has become a widely employed tool in several research studies. Conversely, inadequate breeding and genotyping without considering the potential for non-specific Cre-recombinase expression may lead to misinterpretations of results. Nestin-Cre transgenic mice, widely used for the selective deletion of genes in neurons, have been observed to have an incidence of Cre-line germline recombination. In this study, we attempted to generate neuron-specific Glucagon-like peptide 1 receptor (Glp1r) knock-out mice by crossing mice harboring the Nestin-Cre transgene with mice harboring the Glp1r gene modified with loxP insertion, in order to elucidate the role of Glp1r signaling in the nervous system. Surprisingly, during this breeding process, we discovered that the null allele emerged in the offspring irrespective of the presence or absence of the Nestin-Cre transgene, with a high probability of occurrence (93.6%). To elucidate the cause of this null allele, we conducted breeding experiments between mice carrying the heterozygous Glp1r null allele but lacking the Nestin-Cre transgene. We confirmed that the null allele was inherited by the offspring independently of the Nestin-Cre transgene. Furthermore, we assessed the gene expression, protein expression, and phenotype of mice carrying the homozygous Glp1r null allele generated from the aforementioned breeding, thereby confirming that the null allele indeed caused a global knock-out of Glp1r. These findings suggest that the null allele in the NestinCre-Glp1r floxed breeding arose due to germline recombination. Moreover, we demonstrated the possibility that germline recombination may occur not only during the spermatogenesis at testis but also during epididymal sperm maturation. The striking frequency of germline recombination in the Nestin-Cre driver underscores the necessity for caution when implementing precise breeding strategies and employing suitable genotyping methods.

Project description:Regeneration of muscle in the damaged myocardium is a major objective of cardiovascular research, for which purpose many investigators utilize mice containing transgenes encoding Cre recombinase to recombine loxP-flanked target genes. An unfortunate side effect of the Cre-loxP model is the propensity of Cre recombinase to inflict off-target DNA damage, which has been documented in various eukaryotic cell types including cardiomyocytes (CMs). In the heart, reported effects of Cre recombinase include contractile dysfunction, fibrosis, cellular infiltration and induction of the DNA damage response (DDR). During experiments on adult mice containing a widely used Myh6-merCremer transgene, the protein product of which is activated by tamoxifen, we observed large, transient, off-target effects of merCremer, some of which have not previously been reported. On Day 3 after the first of three daily tamoxifen injections, immunofluorescent microscopy of heart sections revealed that the presence of merCremer protein in myonuclei was nearly uniform, thereafter diminishing to near extinction by Day 6; during this time, cardiac function was depressed as determined by echocardiography. On Day 5, peaks of apoptosis and expression of DDR-regulatory genes were observed, highlighted by >25-fold increased expression of Brca1 Concomitantly, the expression of genes encoding cyclin-A2, cyclin-B2 and cyclin-dependent kinase 1, which regulate the G2/S cell-cycle transition, were dramatically increased (>50- to 100-fold). Importantly, immunofluorescent staining revealed that this was accompanied by peaks in Ki67, 5'-bromodeoxyuridine and phosphohistone H3 labeling in non-CMs, as well as CMs. We further document that tamoxifen-induced activation of merCremer exacerbates cardiac dysfunction following myocardial infarction. These findings, when considered in the context of previous reports, indicate that the presence of merCremer in the nucleus induces DNA damage and unscheduled cell-cycle activation. Although these effects are transient, the inclusion of appropriate controls, coupled with an awareness of the defects caused by Cre recombinase, are required to avoid misinterpreting results when using Cre-loxP models for cardiac regeneration studies.This article has an associated First Person interview with the first author of the paper.

Dataset Information

Myh6 promoter-driven Cre recombinase excises floxed DNA fragments in a subset of male germline cells.

Publications

Myh6 promoter-driven Cre recombinase excises floxed DNA fragments in a subset of male germline cells.

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