pTRIPZ lentiviral vector induction - (Jun/10/2013 )

Hi,

I have recently used the pTRIPZ vector from Thermoscientific and I have encountered some problem that I really hope if anyone could help me out with it.

I infected several cell lines of my interest with the pTRIPZ vector and all cell lines have survived the puromycin selection (2ug/ml), suggesting the vector has successfully been integrated into the genome of infected cell lines. However, when I add the doxycycline, only one cell line showed the fluorescence signal under the microscope and the rest of the cell lines have absolutely no induction. This doesn't make any sense to me. I have tried a couple of times already and even re-infected the cell lines with much higher titer pTRIPZ virus by concentrating the virus, but I still do not get any induction upon adding doxycycline.

I would really appreciate it if anyone could help me out. Thanks.

I am wondering the concentration of doxycycline you used for induction. Maybe you can try a higher Doxy concentration, like 2 μg/mL doxycycline as long as your cell of interest can survive this.
One more thing, observe the expression of RFP with fluorescence microscope at 48 hours after doxycycline addition.