Luciferase-tagged Glioma Stem Cell Model: A Lentivirus-mediated Method to Prepare Luciferase-tagged Glioma Stem Cells for In Vitro Therapeutic Studies
Transcription
Glioma stem cells or GSCs are a subpopulation of glioma – a cancer of glial cells in the brain.
Tagging these GSCs with bioluminescent enzymes like luciferase helps establish high throughput imaging assays for drug screening.
To prepare luciferase tagged GSCs, begin with a culture of GSCs in a suitable growth medium.
Centrifuge the culture to obtain a cell pellet and remove the supernatant.
Add suitable digestive enzymes to the pellet. These enzymes dissociate cell clusters and degrade any traces of connective tissue to obtain a single-cell suspension.
Now, centrifuge the single-cell suspension and remove the supernatant. Resuspend the pellet in a fresh medium.
Next, seed the desired concentration of cells in a culture plate.
Incubate the culture and allow the cells to adhere to the culture plate.
Subsequently, add the desired titer of a virus-containing-gene construct for luciferase-enhanced green fluorescent protein or EGFPand incubate.
The virus interacts with the surface receptors of GSCs, triggering the release of viral RNA containing luciferase-EGFP. Cellular enzymes catalyze reverse transcription of RNA to double-stranded DNA and integrate it into the genome of GSCs.
When visualized under a fluorescence microscope, luciferase-expressing cells appear green due to the fluorescence emitted by EGFP.
