Use of the Real Time Polymerase Chain Reaction Method to Establish a Tightly Regulated Ecdysone Inducible System in Mammalian Cells

Abstract

Introduction: The ecdysone inducible system potentially allows the study of conditional expression of the ex- ogenous reporter genes that may be cell lethal or alter the phenotype during the selection of transfectants. The system re- lies on two independent transfections of plasmids named pVgRXR and pIND. Disruption of the regulatory element within the plasmid during stable integration can result in silenced or high background expression of the exogenous reporter gene. Previous studies (1) have reported a transient luciferase reporter assay to screen the cell lines stably transfected with pVgRXR plasmid. However, there is no suitable method to screen the subsequent pIND transfection. In this study, we demonstrate a real time polymerase chain reaction (PCR) strategy to screen for background expression problems associ- ated with the ecdysone expression system and to simultaneously allow discrimination between the products of endoge- nously expressed and transfected genes. Method: Two screening methods were applied sequentially in order to establish a functional ecdysone expression system. Firstly, the HCT116/VgRXR#8 (a human colon cancer cell line stably transfected with pVGRXR plasmid) was estab- lished by utilising the previously reported luciferase reporter assay (1). Finally, the functional ecdysone system (HCT116/VgRXR#8/mutant p53) was established by a real time PCR screening strategy. This PCR based screening method for the exogenous reporter gene was made possible by utilizing the unique BGH polyA tail from the exogenous reporter gene. Result: Even when the same parental cell line (HCT116/VgRXR#8) was used in the subsequent transfection, background expression was still a common phenomenon. This can be monitored by efficient and sensitive real time PCR. Further- more, the primers designed in this study have high specificity for the exogenous reporter gene. Conclusion: The combined use of luciferase and real time PCR methods was necessary to enable the establishment of a tightly regulated ecdysone inducible system in mammalian cells.