In vivo pharmaceutical target screening using a lentiviral inducible-knockdown shRNA system


Session type:

Yinfei Yin1, Rajendra Kumari1, Sue Watson1, Brett Hall2, Martin Page2, Peter King2, Anna Grabowska1
1Ex Vivo Cancer Pharmacology Centre Of Excellence, Division Of Pre-Clinical Oncology, University Of Nottingham, Nottingham, United Kingdom,2OrthoBiotech, Oncology Research & Development, Beerse, Belgium


Small interfering RNAs (shRNAs) are able to suppress expression of essentially any gene through the endogenous cellular process of RNA interference pathway and have been used in many studies to screen for therapeutic targets in various pathological conditions. In this project, we aimed to develop an efficient in vivo cancer target validation method using lentiviral inducible-knockdown shRNA. To validate our approach, we used the cell-cycle protein polo-like kinase 1 (PLK1) as a proof of concept target.


An inducible shRNA construct targeting PLK1 or a control construct was transfected into colon carcinoma cell line SW620. PLK1 knock-down was quantified by real-time PCR. Inducible shRNA expression (marked by Turbo RFP) was monitored by fluorescence microscopy and flow cytometry. Xenografts were established by subcutaneous or orthotopic injection of PLK1 inducible-knockdown cells into nude mice. Therapeutic effect was assessed by calliper measurement of tumour size.


Following doxycycline induction, PLK1 inducible-knockdown cells showed dose- and time-dependent PLK1 down-regulation, which was consistent with induction of shRNA expression. There was about 60% gene knock-down and 40% protein knock-down 72 hours post-induction. In the in vivo model, there was a decline in tumour growth rate in the PLK1 knockdown group compared with the control group. Ex vivo analysis showed significantly lower PLK1 gene and protein expression in the doxycycline-treated group compared with the control group.


The results support the anti-tumour effects of PLK1 down-regulation and confirm an efficient methodology for cancer target screening using a lentiviral inducible-knockdown shRNA system