Without Doxycycline (Dox), neither shRNA nor ectopic GOI expression takes place and the endogenous wild-type protein is present in the cell. In contrast, with Dox, wild-type transcripts are degraded by the expressed shRNA, while the ectopic protein is present.
The validity of VariCHECK has been successfully demonstrated for the functional analysis of an oncology target (GOIx). A stable cell line was generated that expressed the TET-inducible shRNA against GOIx, previously validated with RNAiONE. Gene knockdown was quantified, and the best performer cell clone with near quantitative knockdown was then transduced with lentiviral vector 2 that encoded the inducible ectopic undegradable GOIx.
Expression of GOIx in the absence and in the presence of Dox was quantified by real-time PCR and Western blot analysis, respectively, confirming an almost quantitative switch from wild type to an ectopically expressed form of GOIx on mRNA as well as protein level. Moreover, the inhibitory effect of GOIx knockdown on cell proliferation was fully rescued by ectopic expression of the undegradable GOIx, demonstrating the reduced proliferation as a real on-target phenotype (Figures 3A–C).