Dataset Information

Promoter Cre-Specific Genotyping Assays for Authentication of Cre-Driver Mouse Lines.

ABSTRACT: The Cre-LoxP system gene knockout (KO) technology provides cell- and time-specificity of gene ablation to investigate cell-autonomous gene function in vivo, and is paramount for understanding the function of genes involved in bone development, remodeling, and repair. This approach permits gene ablation in a cell- or tissue-specific, differentiation stage-specific, and inducible manner, thanks to the use of well-chosen promoters that drive expression of the Cre recombinase in selected cells/tissues. The generation of these powerful tools has led to the expansion of Cre mouse lines available to the research community, which are often shared within and between laboratories. Although convenient and commonly used, genotyping these Cre lines with a generic set of primers that amplifies the Cre transgene does not distinguish between various Cre-deleter lines. This practice poses the significant risk of mistakenly swapping Cre lineages, as laboratories often host and handle several lines at a time and utilize multiple lines per project. In line with the NIH-led effort to promote authentication of biological reagents and increase scientific rigor, we report here strategies for designing appropriate sets of primers able to discriminate some of most widely used Cre-deleter mouse lines in the field of bone biology, and the validation of 24 of them.

SUBMITTER: Couasnay G

PROVIDER: S-EPMC6478581 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Promoter Cre-Specific Genotyping Assays for Authentication of Cre-Driver Mouse Lines.

Couasnay Greig G Frey Christopher C Elefteriou Florent F

JBMR plus 20190118 4

The Cre-LoxP system gene knockout (KO) technology provides cell- and time-specificity of gene ablation to investigate cell-autonomous gene function <i>in vivo</i>, and is paramount for understanding the function of genes involved in bone development, remodeling, and repair. This approach permits gene ablation in a cell- or tissue-specific, differentiation stage-specific, and inducible manner, thanks to the use of well-chosen promoters that drive expression of the Cre recombinase in selected cell ...[more]

PMID: 31044186

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Project description:Current genetic tools designed to target macrophages in vivo often target cells from all myeloid lineages. Therefore, we sought to generate a novel transgenic mouse which has a tamoxifen inducible Cre-recombinase under the control of the human CD68 promoter (hCD68-CreERT2). To test the efficiency and specificity of the of Cre-recombinase activity we crossed the hCD68-CreERT2 mice with a loxP-flanked STOP cassette red fluorescent protein variant (tdTomato) mouse. We established that orally dosing mice with 2 mg of tamoxifen for 5 consecutive days followed by a 5-day induction period resulted in robust expression of tdTomato in CD11b+ F4/80+ tissue resident macrophages. Using this induction protocol, we demonstrated tdTomato expression within peritoneal, liver and spleen macrophages and blood Ly6Clow monocytes. Importantly there was limited or no inducible tdTomato expression within other myeloid cells (neutrophils, monocytes, dendritic cells and eosinophils), T cells (CD4+ and CD8+) and B cells (CD19+). We also demonstrated that the level of tdTomato expression can be used as a marker to identify different populations of peritoneal and liver macrophages. We next assessed the longevity of tdTomato expression in peritoneal macrophages, liver and splenic macrophages and demonstrated high levels of tdTomato expression as long as 6 weeks after the last tamoxifen dose. Importantly, hCD68-CreERT2 expression is more restricted than that of LysM-Cre which has significant expression in major myeloid cell types (monocytes and neutrophils). To demonstrate the utility of this novel macrophage-specific Cre driver line we demonstrated tdTomato expression in recruited CD11b+CD64+F4/80+ monocyte-derived macrophages within the atherosclerotic lesions of AAV8-mPCSK9 treated mice, with limited expression in recruited neutrophils. In developing this new hCD68-CreERT2 mouse we have a tool that allows us to target tissue resident macrophages, with the advantage of not targeting other myeloid cells namely neutrophils and inflammatory monocytes.

Dataset Information

Promoter Cre-Specific Genotyping Assays for Authentication of Cre-Driver Mouse Lines.

Publications

Promoter Cre-Specific Genotyping Assays for Authentication of Cre-Driver Mouse Lines.

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