A Lentiviral Transcriptional Reporter System to Generate a Glioblastoma Stem Cell Differentiation Reporter Line
Transcription
Begin with glioblastoma stem cells, or GSCs.
Add the lentivirus reporter encoding a green fluorescent protein or GFP driven by the GFAP promoter.
Introduce a cationic polymer.
The polymer neutralizes the negative charges on the virus and GSC surface, enabling viral-host cell membrane fusion and RNA release.
The viral RNA is reverse-transcribed into DNA and integrated into the host genome.
Collect the cells, centrifuge, and discard the supernatant.
Add fresh media and re-plate the cells. Incubate.
The GSCs that differentiate into astroglia express transcription factors that activate the GFAP promoter, inducing GFP expression.
Undifferentiated GSCs fail to express GFP.
The GSCs expand and aggregate to form neurospheres. Collect the neurospheres, centrifuge, and discard the supernatant.
Add enzymes to dissociate the cells. Pipette repeatedly to obtain a single-cell suspension.
Add buffer and transfer the cells to a multi-well plate.
Using flow cytometry, detect the GFP-expressing cells.
