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

- 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 t