Aldh1l1-cre/ERT2 promoter drives flox-mediated recombination in peripheral immune cells in addition to astrocytes (P2-3.007)

Abstract

<h3>Objective:</h3> To characterize the cell specificity of <i>Aldh1l1-cre/ERT2-</i>driven recombination in the spleen and spinal cord of mice during peak experimental autoimmune encephalomyelitis (EAE). <h3>Background:</h3> Transgenic mouse models are deployed to study astrocyte-specific alterations in gene expression in <i>in vivo</i> models of neuroinflammation, stroke and neurodegenerative disease. The widely used <i>Aldh1L1-cre/ERT2</i> promoter demonstrates a high level of astrocyte specificity within the CNS though specificity outside the CNS has not yet been well characterized. <h3>Design/Methods:</h3> We compared the astrocyte specificity of two promoter lines, <i>Tg(Aldh1l1-cre/ERT2)1Khakh</i> and <i>Tg(Gfap-cre)73.12Mvs</i>, by crossing them to the <i>Gt(ROSA)26Sor</i> line, which contains a tdTomato sequence preceded by a <i>loxP</i>-flanked STOP cassette. Heterozygous mice (<i>Aldh1l1-cre/ERT2:ROSA</i> and <i>Gfap-cre</i>:<i>ROSA</i>) express promoter-driven tdTomato. Aldh1l1-cre/ERT2 was activated 2 weeks prior to EAE induction using 5 daily intraperitoneal (ip) injections of 100mg/kg tamoxifen in corn oil (20mg/ml). GFAP-cre is constitutive and does not require activation. EAE was induced at 8–10 postnatal weeks (<i>Aldh1l1-cre/ERT2:ROSA</i> and <i>Gfap-cre</i>:<i>ROSA</i>, n=3 each) with one dorsal lumbar subcutaneous injection of MOG_35–55 + Complete Freund’s Adjuvant (CFA) and two ip injections of 60ng pertussis (PTX) toxin at 2–5 and 20–25 hours. Spleen and spinal cords were processed for flow cytometry at 5 days from disease onset with spleens from healthy <i>Aldh1l1-cre/ERT2:ROSA</i> mice (n=3) as a control. Cells were stained for surface markers for neutrophils, macrophages, B cells and T cells and flow cytometry was measured on a Cytek Aurora Flow Cytometer and analyzed with FCS Express software (De Novo). <h3>Results:</h3> Multiple subpopulations of peripheral immune cells, including neutrophils, CD19⁺ B cells, CD4⁺ and CD8⁺ T cells, expressed <i>Aldh1l1-cre/ERT2</i> driven tdTomato. TdTomato positive immune cell populations were not present in <i>mGFAP-Cre tdTomato</i> mice. <h3>Conclusions:</h3> Aldh1l1-cre/ERT2 induces recombination in peripheral immune cells in addition to astrocytes. Gfap-cre does not and may therefore represent a more specific tool to target astrocytes in neuroinflammatory disease. <b>Disclosure:</b> Mario Amatruda has received personal compensation in the range of $50,000-$99,999 for serving as a Postdoctoral Fellow with The Moun Sinai. Mr. Villavicencio has nothing to disclose. The institution of Dr. Britton has received research support from Crohn’s and Colitis Foundation of America. Dr. Britton has received intellectual property interests from a discovery or technology relating to health care. The institution of Dr. Horng has received research support from National Institutes of Health . The institution of Dr. Horng has received research support from Jayne and Harvey Beker Foundation .