I am using a lentiviral TET-ON system for conditional overexpression of my gene of interest in primary kidney cancer cells. I use viral load from transfection of HEK293T cells after 48 and 72 hours and perform selection for 2 weeks using Puromycin (using a concentration I have optimized for the cells I am using). After to weeks, I use Doxycycline at 2mg/ml (adding fresh media everyday) for 72 hrs. However, in western blots, I only see slight increase in expression of my gene of interest compared to -Dox. How can I increase the efficiency of overexpression?
What is your driver (promoter) for rtTA? Do all of your cells have that or is it built into your plasmid? Hence, what is the design of your plasmid? Do you add rtTA separately via another transduction?
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