Genetic Modification of Primary Human Keratinocytes: A Method for Genetically Manipulating Keratinocytes Using Recombinant Retroviruses
Transkript
– Keratinocytes are the prominent cell type present in the epidermis or the outermost layer of the skin. To genetically modify keratinocytes, begin with a culture of retroviral packaging phoenix cells. Subsequently, add a mixture containing retroviral vectors carrying the gene of interest and a transfection reagent. Incubate the plate for the desired time duration.
The transfection reagent facilitates the delivery of viral genetic material into the packaging cells. These specifically designed cells encode proteins required for retroviral capsid production and promote the packaging of retroviral vector RNA carrying the gene of interest. Upon packaging, mature viruses exit the cells into the media. Now, transfer the virus-suspended media into a fresh culture dish containing primary keratinocytes. Augment the culture with a desired cationic polymer solution.
The cationic polymer neutralizes the negative charges on the viral and keratinocyte membranes, increasing the efficiency of viral fusion with the cell surface. Upon fusion, the virus releases its RNA into the cell cytoplasm, which reverse transcribes into double-stranded DNA. This viral DNA enters the nucleus and integrates with the keratinocyte genome, leading to genetic alterations. In the following protocol, we will show the genetic modification of primary human keratinocytes using retroviral transfection.
– The day before transfection, seed phoenix cells in complete media into a 6-well plate at a density of 800,000 cells per well. On the day of transfection, mix 6 microliters of transfection reagent with 100 microliters of DMEM for each well to be transfected and incubate at room temperature for 5 minutes. Add the mixture to a tube containing 3 micrograms of retroviral vector for each well to be transfected and incubate at room temperature for 30 minutes. Add this entire mixture drop-wise to each well of the phoenix cells and incubate the cells overnight.
The day after transfection, remove the media from the phoenix cells and add 2 milliliters of fresh complete media. On the same day, resuspend primary human keratinocytes in keratinocyte serum free medium with antibiotics and seed into 6-well plates at a density of 75,000 cells per well.
The following day, harvest the media, which now contains viral particles from the transfected phoenix cells. Pass the media through 0.45 micron filter using a syringe to remove any contaminating cells. Add 5 micrograms per milliliter of hexadimethrine bromide to the filtered virus containing media to help mediate the infection process.
Then, pipette 2 milliliters of the virus onto the keratinocytes plated the previous day. Next, spin the 6-well plates in a centrifuge at 200 x g for 1 hour at room temperature. After the spin, remove and discard the media containing virus. Wash the cells once with 1x PBS and then add fresh KCSFM. The following day, infect the same batch of keratinocytes with virus using the procedure just shown.
