June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
An efficient RPE-selective tamoxifen inducible Cre driver mouse line
Author Affiliations & Notes
  • Ming Chen
    Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
  • Lily Kim
    Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
  • Carolyn Lu
    Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
  • Hong Zeng
    Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California, United States
    Transgenic, Knockout, and Tumor Model Center, Stanford University School of Medicine, Stanford, California, United States
  • Douglas Vollrath
    Department of Genetics, Stanford University School of Medicine, Stanford, California, United States
  • Footnotes
    Commercial Relationships   Ming Chen, None; Lily Kim, None; Carolyn Lu, None; Hong Zeng, None; Douglas Vollrath, None
  • Footnotes
    Support  1195480-100-PAIPL
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4470. doi:
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    • Get Citation

      Ming Chen, Lily Kim, Carolyn Lu, Hong Zeng, Douglas Vollrath; An efficient RPE-selective tamoxifen inducible Cre driver mouse line. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4470.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Cre-mediated modulation of gene function in the murine retinal pigment epithelium (RPE) has been widely used to investigate the biology of this important cell layer and to model human diseases. Current postnatal RPE-selective Cre driver lines suffer from limited Cre recombination efficiency and/or mosaic expression. We sought to generate a transgenic mouse line with highly efficient RPE-selective Cre expression that could be temporally regulated. We hypothesized that integration of a BEST1-CreERT2 transgene into the Rosa26 locus would achieve this goal.

Methods : By using phiC31 integrase, a DNA construct consisting of a human BEST1 promoter fragment driving expression of a Cre-estrogen receptor gene fusion (BEST1-CreERT2) was integrated into the Rosa26 locus of C57BL/6 mice. A transcriptional stop signal was placed upstream of the BEST1 promoter to reduce potential interference from the endogenous Rosa26 promoter. Germline transmission was confirmed and Rosa26BEST1-CreERT2 mice were bred with a tdTomato reporter line to produce Rosa26BEST1-CreERT2/CAGGS-loxP-Stop-loxP-tdTomato animals. These mice were given intraperitoneal injections of 4-hydroxytamoxifen (4-OHT) (30 mg/kg) or vehicle for 3 or 4 consecutive days beginning at P14 or at 7 weeks of age. RPE and neural retinal flatmounts and cryosections from various tissues were imaged by fluorescence microscopy. Recombination efficiency was quantified as the mean proportion (± S.E.) of RPE flatmounts with tdTomato expression. Muller glia were quantified by staining retinal flatmounts with antibodies against SOX9 or glutamine synthetase and counting tdTomato positive cells in 8 areas including central and peripheral on superior/inferior and nasal/temporal axes.

Results : Male mice injected with 4-OHT for 4 consecutive days exhibited highly efficient recombination (90.7±4.5% at P14 and 87.3±5.0% at 7 weeks), as did female mice with 4 injections beginning at P14 (84.7±3.2%) (N=3 mice per group, one eye per animal). Less than 0.3% of Muller glia also exhibited 4-OHT dependent tdTomato expression, but no tdTomato fluorescence was observed in other ocular cells or multiple tissues.

Conclusions : We have generated a mouse line with highly efficient postnatal induction of Cre-mediated recombination in the RPE and a small fraction of Muller glia. This line will speed efforts to create and study mice with RPE-selective gain and loss of gene function.

This is a 2020 ARVO Annual Meeting abstract.

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