Stmc-29. genetic engineering of glioma cells with histone-2b-gfp tag to track and characterize quiescent glioma stem cells

Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis and limited therapy options. Advances in culture methods have led to the establishment of patient-derived glioma stem cell (GSC) lines that preserve genomic and physiologic characteristics of GBM. Quiescent populations of GSCs have been proposed as the main source of GBM recurrence after therapy. We are applying CRISPR to introduce a doxycycline inducible histone-2B-GFP (H2B-GFP) labeling system at the AAVS1 locus, a "safe harbor" for transgene insertions. The H2B-GFP label allows to track and isolate quiescent glioma cells that retain high GFP label (dividing cells dilute H2B-GFP), and to characterize their cancer stem cell properties. We have transfected GSC lines with an AAVS1 Cas9 plasmid and a targeting plasmid carrying Tet-ON transactivator M2rtTA and tetO-H2B-GFP. Cells with integrated targeting vector were selected with G418, and insertions were confirmed by PCR over 5’ and 3’ arms. H2B-GFP GSC lines were transplanted intracranially into the striatum of SCID mice, and after an initial pulse period of 2 weeks (doxycycline-ON), tumors are allowed to expand during chase periods of 2, 4, or 8 weeks. Subcohorts of mice are subjected to radiation therapy to elucidate the contribution of quiescent GSC to radiation resistance. We are using histology to reveal the quiescent cell populations (GFP-high) in tumors, their spatial distribution and proximity to vascular niches, and their ...
Source: Neuro-Oncology - Category: Cancer & Oncology Authors: Tags: STEM CELLS Source Type: research