Establishment Of Cells Stably Expressing Luciferase Containing Nonsense Mutation

Objective: To construct cells stably expressing luciferase containing nonsense mutation for rapid screen of compounds which can induce readthrough of nonsense mutations.Methods: Plasmids pGL4-WT and pGL4-MUT were digested by endonuclease firstly,wild type luciferase cDNA and nonsense mutant were then inserted into the multiple cloning site of lentiviral vector pLVX-IRES-Neo respectively.After identification by restriction analysis and PCR,the recombinant lentiviral vectors were packaged into lentiviral particles for infection of HEK293 cells.Stably infected HEK293 cells were selected and identified by reverse transcription PCR.Finally,stably infected HEK293 cells were treated with G418,a positive nonsense mutation readthrough agent,and the protein level and activity of luciferase were detected using Western blot and chemiluminescence assay respectively.Results: After digestion,2.7 kb wild type luciferase cDNA and nonsense mutant were both obtained.The recombinant lentiviral vectors pLVX-WT and pLVX-MUT were successfully constructed,confirmed by the results of Eco R? digestion,Nhe?and Bam H? digestion,as well as PCR.The result of reverse transcription PCR of luciferase demonstrated that HEK293 cells stably infected with pLVX-WT and pLVX-MUT were successfully selected,which named as HEK293 WT and HEK293 MUT respectively.It was found that HEK293 WT cells stably expressed luciferase and showed the same luciferase activities treated with G418 or not.HEK293 MUT cells did not express luciferase or show any luciferase activity before G418 treatment.After G418 treatment,HEK293 MUT cells expressed luciferase which activity is equal to 20% of that in HEK293 WT.Conclusion: HEK293 cells stably expressing luciferase containing nonsense mutations were successfully established and may be helpful for screening new compounds which can induce readthrough of nonsense mutations.