This study investigates the transduction of glioblastoma stem cells (GSCs) using a lentivirus encoding green fluorescent protein (GFP). The methodology involves the use of a cationic polymer to facilitate viral entry and subsequent expression of GFP in differentiated GSCs.
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.
To begin seed 1 million glioblastoma stem cells or GSCs in 2 milliliters of complete neural stem cell growth medium in a 6-well plate. Next, transduce the cells with the lentivirus reporter at a multiplicity of infection equal to five.
To the cell and virus mixture, add 2 microliters of polybrene such that the final concentration is 8 micrograms per milliliter, and incubate the cells at 37 degrees Celsius and 5% CO2 overnight. After incubation, replace the growth medium to remove unbound virus, and centrifuge cells at 360 times g for 5 minutes. Then, carefully aspirate medium and plate cells back in a 6-well plate, and continue to incubate the cells for 24 hours.
Harvest 0.5 milliliters of the total culture volume. Pellet cells by centrifugation at 360 times g for 5 minutes at room temperature. Resuspend cells in 0.5 milliliters fresh neural stem cell growth medium.
Place the plate back in the incubator. To allow for expansion for at least 72 hours. Add 200 microliters of dissociation reagent, and incubate for five minutes in a water bath set to 37 degrees Celsius. Dissociate cells by pipetting up and down gently. To the dissociated cells, add 800 microliters of HBSS to ensure minimal cell attachment or aggregation.
Next, transfer 200 microliters of each cell suspension into wells of a 96 multi-well plate. Determine the percentage of cells expressing the green fluorescent protein by flow cytometry. Record at least 10,000 viable cells in each acquisition.
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