Lentiviral Transduction and Bioluminescent Imaging

Third generation lentiviral particles were produced by transient co-transfection of 293FT cells with Akaluc^{54 (link)}-plasmid (Kindly provided by Prof. Greg Hannon) and the three packaging constructs pMDL, CMV-Rev, and VSV-G. Lentivirus containing particles were concentrated on Centricon Plus-70 filters (Millipore) and B16.F10 cells were infected with concentrated lentivirus in the presence of 8 mg/ml polybene, and selected 3 days after infection with neomycin (Geneticin 1 mg/ml, Invitrogen). IVIS bioluminescent imaging was performed by in vitro exposure of B16.F10-Akaluc cells to 250 µM Akalumine substrate for 30 minutes at 37 °C in a humidified, 5% CO₂ incubator. The cells were washed with PBS twice and 1×10^{5 (link)} cells were injected i.v. into B6.Tyr^−/−mice and imaging was performed 2, 10, and 30 min after. Total photon emission from the thorax of each mouse was quantified with the LivingImage software package (Xenogen).