Browsing UMB Open Access Articles by Subject "Iba1"
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Generation of an Iba1-EGFP transgenic rat for the study of microglia in an outbred rodent strainNeuroscience has been transformed by the ability to genetically modify inbred mice, including the ability to express fluorescent markers specific to cell types or activation states. This approach has been put to particularly good effect in the study of the innate immune cells of the brain, microglia. These specialized macrophages are exceedingly small and complex, but also highly motile and mobile. To-date, there have been no tools similar to those in mice available for studying these fundamental cells in the rat brain and we seek to fill that gap with the generation of the genetically modified Sprague-Dawley rat line: SD-Tg(Iba1-EGFP)Mmmc. Using CRISPR-Cas/9 technology, we knocked in EGFP to the promoter of the gene Iba1 With 4 male and 3 female founders confirmed by qPCR analysis to have appropriate and specific insertion, we established a breeding colony with at least 3 generations of backcrosses to obtain stable and reliable Iba1-EGFP expression. Specificity of EGFP expression to microglia was established by flow cytometry for CD45low/CD11b+ cells and by immunohistochemistry. Microglial EGFP expression was detected in neonates and persisted into adulthood. Blood macrophages and monocytes were found to express low levels of EGFP, as expected. Lastly, we show that EGFP expression is suitable for live imaging of microglia processes in acute brain slices and via intravital 2-photon microscopy. SIGNIFICANCE STATEMENT: Neuroscience research in rat models has lagged compared to the mouse due to limitations in the ability to generate genetic modifications. In order to fill part of that gap, we have generated a transgenic rat in which the innate immune cells of the brain, microglia, express EGFP. This modification allows for isolation of microglia from other cells by flow cytometry or FACS for detailed transcriptomic and proteomic analysis. The visualization of EGFP in acute brain slices or by in vivo imaging further enhances the ability to interrogate the role of these critical cells across the lifespan and in health and disease.