Abstract Details

Title Aldh1l1-cre/ERT2 promoter drives flox-mediated recombination in peripheral immune cells in addition to astrocytes

Topic Multiple Sclerosis

Presentation(s) P2 - Poster Session 2 (11:45 AM-12:45 PM)

Poster/Presentation
Number 3-007

Objective To characterize the cell specificity of Aldh1l1-cre/ERT2-driven recombination in the spleen and spinal cord of mice during peak experimental autoimmune encephalomyelitis (EAE).

Background Transgenic mouse models are deployed to study astrocyte-specific alterations in gene expression in in vivo models of neuroinflammation, stroke and neurodegenerative disease. The widely used Aldh1L1-cre/ERT2 promoter demonstrates a high level of astrocyte specificity within the CNS though specificity outside the CNS has not yet been well characterized.

Design/Methods We compared the astrocyte specificity of two promoter lines, Tg(Aldh1l1-cre/ERT2)1Khakh and Tg(Gfap-cre)73.12Mvs, by crossing them to the Gt(ROSA)26Sor line, which contains a tdTomato sequence preceded by a loxP-flanked STOP cassette. Heterozygous mice (Aldh1l1-cre/ERT2:ROSA and Gfap-cre:ROSA) 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 (Aldh1l1-cre/ERT2:ROSA and Gfap-cre:ROSA, 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 Aldh1l1-cre/ERT2:ROSA 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).

Results Multiple subpopulations of peripheral immune cells, including neutrophils, CD19⁺ B cells, CD4⁺ and CD8⁺ T cells, expressed Aldh1l1-cre/ERT2 driven tdTomato. TdTomato positive immune cell populations were not present in mGFAP-Cre tdTomato mice.

Conclusions

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.

Authors/Disclosures
Sam H. Horng, MD, PhD PRESENTER	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 .
Mario Amatruda (Icahn School of Medicine at Mount Sinai)	No disclosure on file
Jorge Villavicencio (Icahn School of Medicine Mount Sinai)	No disclosure on file
	No disclosure on file

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