Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Abstract

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy

“…M7 and OL61 neurospheres were derived from sleeping beauty transposase system-induced tumors in wild-type mice. 44,45 While M7 tumors were generated using NRASG12V and SV40-large T antigen plasmids, OL61 tumors contained NRASG12V, short hairpin (sh)-targeting p53, and platelet-derived growth factor b (PDGFb) overexpression. 44 Almost 80% of the CD45 high compartment was comprised of MDSCs ( Figure S1A).…”

“…44 Neurospheres were derived from tumors generated in C57BL/6 mice using the Sleeping Beauty (SB) transposase system. 44,45 Tumor tissue was dissociated using HyQTase (HyClone), passed through a 70-mm cell strainer, and cultured in media containing DMEM-F12, N2, and B27 supplements, 1% penicillin-streptomycin (Invitrogen), Normocin (Invivogen), and supplemented with 20 ng/mL EGF and 20 ng/mL FGF (Peprotech). Growth factors were added every 2 to 3 days, and cells were passaged once to twice each week, depending on density.…”

Survival and Proliferation of Neural Progenitor–Derived Glioblastomas Under Hypoxic Stress is Controlled by a CXCL12/CXCR4 Autocrine-Positive Feedback Mechanism

“…NPC-derived tumors were generated and characterized by histology and immunohistochemistry as previously described (31) and elaborated in the Supplementary Methods. In vivo treatment with AMD3100 is detailed in the Supplementary Methods.…”

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

“…Induce the brain tumor in mouse using the Sleeping Beauty transposon system in which plasmids encoding oncogenes or shRNA targeting tumor suppressors is injected into the lateral ventricles of neonates to generate spontaneous brain tumors 9 . Select a mouse with a large tumor, which is confirmed with bioluminescence imaging.…”

“…NOTE: More detailed information about the Sleeping Beauty Transposon System and plasmid constructs used can be found in Calinescu et al 9 .…”