Genetic Modification of Primary Human Keratinocytes: A Method for Genetically Manipulating Keratinocytes Using Recombinant Retroviruses

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Genetically Modifying Primary Human Keratinocytes

NOTE: Use amphotropic phoenix cells grown in complete DMEM media [DMEM + 10% fetal bovine serum (FBS) + pen/strep] to produce viruses to infect primary human keratinocytes. Viral packaging genes that encode proteins such as gag-pol and envelope are stably integrated into the phoenix cell genome, which allows for virus production when transfected with a retroviral vector. Phoenix cells can be transfected with high efficiency allowing for high viral titer production. Viruses produced from this packaging line can also infect a large variety of mammalian cells including humans.

1. Day 1: The day before transfection, seed phoenix cells in a 6-well plate at a density of 800,000 cells per well in complete DMEM media.
2. Day 2: The day of transfection, use 3 μg of retroviral vectors per well. Aliquot 3 μg of retroviral vector into a 1.5 mL tube. Use a mix of 100 μL DMEM plus 6 μL of transfection reagent for every 3 μg of vector. Mix 6 μL of transfection reagent with 100 μL DMEM in a 1.5 mL tube. (The retroviral vectors that are used are listed in the equipment/materials table)
   1. Incubate for 5 min and add the 106 μL mixture into the pre-aliquoted tube containing 3 μg of retroviral vector. Mix and incubate at RT for 30 min. Add this entire mixture dropwise to each well of the phoenix cells.
3. Day 3: The day after transfection, remove the media from the phoenix cells and add 2 mL of fresh complete DMEM media. On the same day, seed primary human keratinocytes into 6-well plates at a density of 75,000 cells per well. Maintain the keratinocytes in a keratinocyte serum free medium (KCSFM) with antibiotics (pen/strep).
   NOTE: Primary human keratinocytes were purchased. Cells can be purchased from a variety of vendors (listed in the Materials table).
4. Day 4: Harvest the media from the transfected phoenix cells, which now contain viral particles. Pass the media through a 0.45 μm filter using a syringe (to remove any contaminating phoenix cells) and place 2 mL onto the keratinocytes (plated at 75,000 cells/well the previous day). Add hexadimethrine bromide (5 μg/mL) to the media to help mediate the infection process. Viral titers are ~1 x 10⁷ TU/mL.
   1. Spin the 6-well plates in a centrifuge at 200 x g for 1 hr at RT. After the spin, remove the media containing virus and wash the cells once with 1x PBS before adding KCSFM.
5. Day 5: Infect the same batch of keratinocytes again as in 1.4, using 2 mL of filtered media containing hexadimethrine bromide (5 μg/mL). Spin it at 200 x g for 1 hr at RT to remove media containing virus and wash with 1x PBS then add KCSFM.
   NOTE: Select the keratinocytes using a drug if there is a selectable marker on the retroviral vector and expand for use in downstream analysis or applications such as organotypic cultures.