Genetically Engineered and Implantable Mouse Brain Tumor Models: Characterization by Immunohistochemistry and Flow Cytometry.

Gliomas are aggressive tumors with a poor prognosis. The protocols presented here outline the methods used to study tumor progression, the tumor microenvironment (TME), and the effects of experimental treatments. The Sleeping Beauty (SB) transposase system induces tumors de novo to generate mouse models that recapitulate human gliomas. Plasmids are constructed with oncogenic drivers and other genetic alterations of interest. which are recognized by their unique position in between inverted/direct repeat (IR/DR) sequences. Luciferase enzyme is used to monitor the uptake of the plasmid, tumor growth, and response to experimental therapies. The genes of interest are tracked using fluorescent markers. Tumors will arise in immunocompetent hosts, which provides a relevant preclinical platform for analysis of tumor initiation, progression, survival, immune microenvironment, and histopathological features. Once the tumor grows within the desired brain location, it can be harvested to generate cell cultures of neurospheres for future experimentation. The benefit of implantable models generated from SB tumors is that they provide specific anatomical and genetic context, in which specific genetic characteristics can be tracked, as they are co-expressed with fluorescent markers. Post glioma cell implantation, additional analysis of the TME and tumor growth can be performed through immunohistochemistry (IHC) and flow cytometry. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Creation of mouse glioma models by Sleeping-Beauty-mediated transposition Basic Protocol 2: Generation of orthotopic implantable brain tumors and neurospheres Basic Protocol 3: Hematoxylin and eosin staining of glioma tissue samples Basic Protocol 4: Immunohistochemistry of glioma tissue samples Basic Protocol 5: Flow cytometry for immune cell analysis of the tumor microenvironment.