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Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in Pten^LoxP/LoxP;Braf^CA/+ Mice.

ABSTRACT: Current genetically-engineered mouse melanoma models are often based on Tyr::CreER^T2-controlled MAPK pathway activation by the BRAF^V600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreER^T2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre recombinase, including injection of lentiviral particles, DNA tattoo administration and particle-mediated gene transfer, to induce melanomas in Pten^LoxP/LoxP;Braf^CA/+ mice lacking the Tyr::CreER^T2 allele. We found that dermal delivery of the Cre recombinase gene under the control of a non-specific CAG promoter induced the formation of melanomas, but also keratoacanthoma and squamous cell carcinomas. Delivery of Cre recombinase DNA under the control of melanocyte-specific promoters in Pten^LoxP/LoxP;Braf^CA/+ mice resulted in sole melanoma induction. The growth rate and histological features of the induced tumors were similar to 4-hydroxytamoxifen-induced tumors in Tyr::CreER^T2;Pten^LoxP/LoxP;Braf^CA/+ mice, while the onset of spontaneous tumors was prevented completely. These novel induction methods will allow long-term experiments in mouse models of skin malignancies.

SUBMITTER: Deken MA

PROVIDER: S-EPMC5187949 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in PtenLoxP/LoxP;BrafCA/+ Mice.

Deken Marcel A MA Song Ji-Ying JY Gadiot Jules J Bins Adriaan D AD Kroon Paula P Verbrugge Inge I Blank Christian U CU

International journal of molecular sciences 20161220 12

Current genetically-engineered mouse melanoma models are often based on Tyr::CreERT2-controlled MAPK pathway activation by the BRAFV600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreERT2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre rec ...[more]

PMID: 27999416

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Project description:The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wild-type strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.

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

Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in Pten^LoxP/LoxP;Braf^CA/+ Mice.

Publications

Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in Pten<sup>LoxP/LoxP</sup>;Braf<sup>CA/+</sup> Mice.

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