Lentiviral transduction, no target protein expression and lost other reporter
Posted 27 July 2011 - 07:37 AM
I ordered a PC-3M-luc-C6 cell line from Caliper Life Sciences, which expresses firefly luciferase. I transduced the cells with renilla luciferase lentiviral particles. I grew up colonies that were resistant to Puromycin. However, when I tested the cells' firefly and renilla luciferase activity, I found that there was no renilla luciferase activity and I had lost the firefly luciferase activity. I'd appreciate any help with regards to what may have happened and how I could fix it.
Posted 12 August 2011 - 11:12 PM
Also... I believe in order for cells to express antibiotic resistance to those selection, for ~1 day after lentiviral particles, incubate cells with no selection (puromycin). However, from what I see from your cells you, I would incubate cells with zeocin in order to keep PC-3M-Luc-C6 cells to express firefly luciferase. Some people don't start selecting until like 2-4days later in order to increase 'their chance'.
Do you have proper positive control for lentiviral particle trasduction efficiency?
You also might have to do a curve for puromycin selection. Make sure to select cells with both, zeocin and puromycin.
Edited by powertoeki, 12 August 2011 - 11:19 PM.