Establishing Oncogenic RAS in Primary Keratinocytes

The v-ras^Ha replication-defective ecotropic retrovirus was prepared using Psi2 producer cells. Retrovirus titers were routinely 1 × 10⁷ virus/mL. Cultured primary keratinocytes were infected with v-ras^Ha retrovirus (here referred to as RAS or oncogenic RAS) on day 3 at a multiplicity of infection (MOI) of 1 in medium containing hexadimethrine bromide (4 μg/mL; Sigma, 107689). The IκBsr (IκB super repressor) adenovirus or Cre recombinase (Cre) were introduced into primary keratinocytes using an adenoviral construct driven by a cytomegalovirus promoter, and a similarly constructed adeno-GFP was used as control. Keratinocytes were adenovirus-infected for 30 minutes in serum-free medium with a MOI of 10 viral particles per cell and hexadimethrine bromide (4 μg/mL) to enhance uptake. Serum-containing medium was added to the cells for the next 48 hours after the infection. The cDNAs for human RAS oncogenes were purchased from Addgene: pBabe HRAS 12V (plasmid #12545), pBabe KRAS 12V (plasmid #12544). The promoter FerH (25 (link)) and RAS cDNAs were cloned into pLV-CE vector and high titer lentivirus (10e⁸ TU per mL) were produced (Cellomics Technology, LLC).