Monitoring ORF10's Impact on Host Transcriptome Using Doxycycline-Inducible A549 and 293T Cell Lines.
To monitor ORF10s effects on the host transcriptome, doxycycline-inducible cell lines were create in A549 and 293T(HEK) cells expressing ORF10. Because doxycycline itself can alter gene expression, we used doxycycline-inducible cell lines driving GFP as control. Empty vectors were used as well. Overall design: ORF10 was cloned into pLVX-EF1alpha-IRES-Purovector, swapped into a TRE construct, and used to generate lentivirus. A549 and 293T cells were grown for four days and then transduced and selected with puromycin to generate doxycycline inducible cell lines. Clones were generated and allowed to grow out and samples were then tested for baseline ORF10 expression and then ORF10 induction with doxycycline. Cell lines that had no to minimal ORF10 expression at baseline and then had robust induction were used for downstream assays. GFP and empty vector was swapped with ORF10 as a conrol. ORF10 without doxycycline is included as well.