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An efficient inducible RPE-Selective cre transgenic mouse line.

ABSTRACT: Cre-mediated modulation of gene function in the murine retinal pigment epithelium (RPE) has been widely used, but current postnatal RPE-selective Cre driver lines suffer from limited recombination efficiency and/or ectopic or mosaic expression. We sought to generate a transgenic mouse line with consistently efficient RPE-selective Cre activity that could be temporally regulated. We used ϕC31 integrase to insert a DNA construct encoding a human BEST1 promoter fragment driving a Cre recombinase estrogen receptor fusion (BEST1-CreERT2) at the Rosa26 locus of C57BL/6J mice. Rosa26^{BEST1-CreERT2} mice were bred with a tdTomato reporter line and to mice with a Cre-conditional allele of Tfam. 4-hydroxytamoxifen or vehicle was delivered by four consecutive daily intraperitoneal injections. TdTomato was robustly expressed in the RPE of mice of both sexes for inductions beginning at P14 (males 90.7 ± 4.5%, females 84.7 ± 3.2%) and at 7 weeks (males 84.3 ± 7.0%, females 82 ± 3.6%). <0.6% of Muller glia also expressed tdTomato, but no tdTomato fluorescence was observed in other ocular cells or in multiple non-ocular tissues, with the exception of sparse foci in the testis. No evidence of retinal toxicity was observed in mice homozygous for the transgene induced beginning at P14 and assessed at 7-10 months. RPE-selective ablation of Tfam beginning at P14 led to reduced retinal thickness at 8 months of age and diminished retinal electrical responses at 12 months, as expected. These findings demonstrate that we have generated a mouse line with consistently efficient, tamoxifen-mediated postnatal induction of Cre recombination in the RPE and a small fraction of Muller glia. This line should be useful for temporally regulated modulation of gene function in the murine RPE.

SUBMITTER: Chen M

PROVIDER: S-EPMC7856207 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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An efficient inducible RPE-Selective cre transgenic mouse line.

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