Retroviral-Mediated Transduction: A Method to Introduce Target Gene into Cancer Cells
筆記録
– Retroviruses are RNA viruses that replicate by using reverse transcriptase to convert their RNA genome to double-stranded DNA and permanently integrate into the host genome. New viral particles exit the cell and can infect other cells. Cancer gene therapy employs retroviral vectors to introduce therapeutic genes into target cells via a technique called transduction to downregulate the mutated gene or eliminate cells.
To obtain high-titer retroviral vectors, transfect the HEK 293 cells with a transfection mixture containing the desired retroviral plasmid, packaging plasmid, and a suitable transfecting agent. Incubate the cells. The transfecting agent facilitates the delivery of both plasmids into the cells.
The retroviral plasmid encodes the viral genome with the target gene while the packaging plasmid encodes the viral envelope proteins. The packaging cells' cellular machinery forms retroviral particles and releases them into the medium. Collect the viral supernatant and centrifuge to remove cells.
Transfer the desired concentration of viral particles and cancer cells to a fibronectin-coated culture plate to facilitate binding viral envelope proteins to cells. Incubate the cells. Collect the cells and perform flow cytometry to analyze the transduction efficiency. This protocol will perform retroviral-mediated transduction of c-Kit-positive mouse bone marrow cells.
– First, combine the retroviral plasmid DNA with the packaging plasmid, calcium chloride, and water in a 1.5 milliliter tube. Then add 500 microliters of HBS to another 1.5 milliliter tube. Add the transfection mixture dropwise to the tube containing HBS while simultaneously mixing with the vortexer set to the lowest setting.
Incubate the transfection mixture at room temperature for 20 to 30 minutes. During the incubation period, add chloroquine to the HEK cells and keep them in the incubator. After adding the transfection mix, incubate the cells for 8 hours in a 37 degrees Celsius, 5% CO2 incubator, changing the cell media by adding 14 milliliters of fresh DMEM 10 media very slowly. Pipetting slowly against the wall of the dish helps to prevent any stripping of HEK cells.
Then incubate the cells overnight in the 37 degrees Celsius, 5% CO2 incubator. Use a 10 milliliter syringe to collect the viral supernatant. Then attach a 0.45 micrometer syringe filter to the syringe. Plunge the viral supernatant into a new collection tube. Collect the viral supernatant approximately 16 hours later.
After this, add 2 milliliters of recombinant human fibronectin fragment in PBS to untreated 6-well culture plates. The next day, use a pipette to remove the fibronectin from the plates. Block the plates with 2% BSA in PBS and incubate the plates at room temperature for 30 minutes.
Use a vacuum to remove BSA and wash the plates with PBS before removing it with the vacuum. After this, immediately add the filtered viral supernatant and centrifuge the plate for two hours. Use a vacuum to remove the supernatant and spin the plate again.
Next, remove the supernatant with a vacuum and wash the viral-coated wells with PBS. Remove the PBS with the vacuum. Add the previously cultured c-Kit+ mouse cells to the viral-coated wells. After centrifugation, incubate the cells at 37 degrees Celsius and 5% carbon dioxide. 48 hours after transduction, pellet the cells via centrifugation and resuspend them in FACS buffer number 1.
