I use SMARTvector Inducible Lentiral shRNA vector, which include 2 promoters: costitutive promoter, which control the expression of puromycin resistence and TET-On 3G transactivator and inducible promoter for the expression of my gene of interest linked to TurboGFP. I infected my cell line following datasheet' instuctions and I selected the infected cells with puromycin. After 48h of induction with Doxycycline I observed that only 40% of infected cells were GFP+. I obtained two different population: GFP+ and GFP-, but GFP- were not inducible cells, even though they "survived" puromycin treatment.
My questions are:
1. Which efficiency of induction can I expect with this system?
2. Why my GFP- infected cells are resistent to the puromycin, but they are not inducible with doxyxycline?
Try seeding a 24 well plate and trying various concentrations of your shRNA vector. You may not be treating the cells with enough plasmid, thus you are only getting the 40% efficiency. Did you concentrate your lentivirus particles?
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