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Generation and Characterization of a Rosa26-CreERT2 Mouse Model for Conditional Gene Ablation in CNS Injury Studies
Location: 70
Mentor: Dr. Damien Pearse
Transgenic mouse models are critical tools for uncovering mechanistic roles of specific genes in central nervous system (CNS) injury and for identifying promising therapeutic targets. Here, we describe the development and genetic characterization of a mouse strain harboring a tamoxifen-inducible Cre recombinase under control of the Rosa26 locus (Rosa26-CreERT2). By crossing Rosa26-CreERT2 mice with a floxed ATE1 allele, we achieve temporally regulated, tissue-selective ATE1 knockout. In our breeding strategy, pups were weaned at three weeks, and tail biopsies were collected for DNA extraction and genotyping via polymerase chain reaction (PCR). Through these analyses, we confirmed the presence of both the Rosa26-CreERT2 transgene and the floxed ATE1 allele in
multiple lines. This approach enables gene deletion to be induced at specific developmental stages or in adult mice, thereby circumventing embryonic lethality or compensatory adaptations often observed in constitutive knockouts.
To evaluate whether recombination occurs in a cell-type-specific fashion relevant to CNS injury, we administered tamoxifen to adult mice and performed spinal cord injury (SCI) at the thoracic (T8) level using the Infinite Horizons impactor. Post-injury tissues were cryosectioned and subjected to immunohistochemistry, leveraging antibodies for markers of motor neurons, microglia, and ATE1. Our preliminary results demonstrated robust Cre-mediated recombination in targeted cell populations, as indicated by the loss of ATE1 immunostaining in tamoxifen-
treated animals. Quantification of ATE1-negative cells revealed a high percentage of knockout in the targeted regions. These findings establish Rosa26-CreERT2 as a flexible and powerful platform for the spatiotemporal control of ATE1 deletion in CNS injury models. Ongoing work will refine this model’s specificity and investigate the downstream functional outcomes of ATE1 ablation in injury and regeneration contexts.
