Sura, Survasha; Department of Biological Sciences; Department of Biochemical and Molecular Biology; Georgia Cancer Center; Rajpurohit, Surendra K; Augusta University (2019-02-13)
      Zebrafish have emerged as a powerful model organism for elucidating the development and function of microglia. Generation of new transgenic reporter lines and imaging tools strengthen the zebrafish model in microglia study�in-vivo. The aim is to develop a novel compound transgenic line to study the inflammatory process mediated by NF-kB in microglia cells. This novel compound transgenic line will establish a new model for microglia study. To generate the novel compound zebrafish transgenic model for microglia, we are crossbreeding microglia transgenic line zebrafish (Tg(mpeg1:mCherry) with the NF-kB Tg(6xNFkB:EGFP) transgenic progeny. We first generate a heterozygous F1 progeny which will be bred to generate an F2 homozygous progeny. Once the F1 progeny of the Microglia-NfkB transgenic line is developed, they will be crossbred to develop the Homozygous compound transgenic line. Fluorescent Microscopy will be used to screen the larvae generated from the breeding events. By developing the compound transgenic line, we are optimizing microglia isolation and sorting methodology by using the related antibodies as the marker. The NF-kB microglia transgenic line will provide a unique platform for drug screening to address microglial based ailments, thus furthering the understanding and treatment of human disease.
    • Isolation and Culture of Microglia

      Doughty, Deanna; Venugopal, Natasha; Department of Biological Sciences; Bradford, Jennifer; Department of Biological Sciences; Augusta University (2018-02-12)
      Glioblastoma (GBM) is the most aggressive and common adult brain tumor subtype, with the majority of patients surviving less than one year. The GBM microenvironment is composed of tumor cells as well as non-cancerous cells, such as microglia, a component of the immune system in the brain. To better understand the role of microglia in GBM, we have optimized in vitroculture conditions for primary microglia. Growing microglia in culture is challenging, but this technique is needed for planned future experiments. Microglia were isolated from mouse neuronal tissue by magnetic bead antibody cell separation using the cellular marker CX3CR1. Isolated microglia were then cultured in various culture conditions, and cellular morphology by light microscopy was used to determine cell health, viability, and activation status. It was determined that the primary microglia grow best in neurobasal media in wells coated with poly-D lysine. Future studies aim to isolate a larger number of cells to allow forco-culture of the inactivated microglia with GBM cells. These results will allow us to better understand the role that microglia play in GBM progression.