Journal of neuroscience methods · 2019
Isolating cells from adult murine brain for validation of cell-type specific cre-mediated deletion.
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Kosior Natalia, Petkau Terri L, Connolly Colúm, Lu Ge, Leavitt Blair R
Аннотация
BACKGROUND: TheCre/loxP system allows for the temporal and spatial investigation of the expression of a single gene in the nervous system. Current methods of validating conditional knock-out mouse models rely on heterogeneous brain tissue or primary culture. These methods may assess the extent of genetic knockdown in the brain but do not provide age-appropriate, cell-type specific information.
NEW METHOD: We isolated specific cell types from adult murine brain using FACS to assess cell type-specific gene expression in conditional mouse models.
RESULTS: We identified robust but incomplete genetic knockdown in microglia isolated from two separate microglia-specific knockout models.
COMPARISONWITH EXISTING METHODS(S): Genetic knockdown in isolated adult microglia differed significantly from cultured primary microglia.
CONCLUSIONS: Differences observed in primary cultured microglia compared to isolated adult microglia suggest that current methods used to validate microglia-specific gene deletion over-estimate deletion efficiency. Assessment of gene expression in isolated adult microglia provides a more accurate assessment of Cre-mediated gene deletion.
NEW METHOD: We isolated specific cell types from adult murine brain using FACS to assess cell type-specific gene expression in conditional mouse models.
RESULTS: We identified robust but incomplete genetic knockdown in microglia isolated from two separate microglia-specific knockout models.
COMPARISONWITH EXISTING METHODS(S): Genetic knockdown in isolated adult microglia differed significantly from cultured primary microglia.
CONCLUSIONS: Differences observed in primary cultured microglia compared to isolated adult microglia suggest that current methods used to validate microglia-specific gene deletion over-estimate deletion efficiency. Assessment of gene expression in isolated adult microglia provides a more accurate assessment of Cre-mediated gene deletion.