Abstract
The use of inducible transgenic Nestin-CreERT2 mice has proved to be an essential research tool for gene targeting and studying the molecular pathways implicated in adult neurogenesis, namely, inside the adult subgranular zone (SGZ) of the dentate gyrus and the adult subventricular zone (SVZ) lining the lateral ventricles. Several lines of Nestin-CreER-expressing mice were generated and used in adult neurogenesis research in the past two decades; however, their suitability for studying neurogenesis in aged mice remains elusive. Here, we assessed the efficiency of Cre-loxP genetic recombination in the aging SVZ using the Nestin-CreERT2/Rosa26YFP line designed by Lagace et al. (J Neurosci 27(46):12623–12629, 2007). This analysis was performed in 12-month-old (middle-aged) mice and 20-month-old (old) mice compared to 2-month-old (young adult) mice. To evaluate successful recombination, our approach relies on the histological assessment of Cre mRNA level of expression and the YFP reporter gene’s expression inside the aging SVZ by combining in situ hybridization and immunohistochemistry. Using co-immunolabeling, this approach also provides the advantage of estimating the percentage of recombined progeny [(GFP+Nestin+)/Nestin+] and the rate of cell proliferation [(GFP+Ki67+)/GFP+] inside the aging SVZ niche.
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Acknowledgments
N.G. laboratory is supported by the University Research Board (URB) at the American University of Beirut, Kamal A. Shair CRSL Research Fund (KAS), Farouk Jabre Biomedical Research Grant (FJ), and the Lebanese National Council for Scientific Research (LNCSR). Part of this study was performed using common equipment and material available at the Kamal A. Shair Central Research Science Laboratory (KAS-CRSL) at AUB. The in situ hybridization protocol described in this study is adopted and modified from Wallace and Raff, Development 1999;126:2901–2909.
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Omais, S., Halaby, N.N., Habashy, K.J., Jaafar, C., Bejjani, A.T., Ghanem, N. (2019). Histological Assessment of Cre-loxP Genetic Recombination in the Aging Subventricular Zone of Nestin-CreERT2/Rosa26YFP Mice. In: Turksen, K. (eds) Stem Cells and Aging . Methods in Molecular Biology, vol 2045. Humana, New York, NY. https://doi.org/10.1007/7651_2019_214
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DOI: https://doi.org/10.1007/7651_2019_214
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