ABSTRACT
@#Objective To develop a duplex digital PCR(dPCR) for evaluation of the stability of luciferase(Luc) gene in Luc2P reporter cell lines(CHO-K1,Hacat,HEK293 and UT7).Methods Genomic DNAs of Luc2P reporter cell lines were extracted,a duplex dPCR was developed to determine the copies of reference gene RPP30 and the target gene Luc,and the relative copy number of Luc(copies Luc/copy RPP30) was employed as the indicator for the stability evaluation of Luc gene;The developed method was verified for the specificity,precision,linearity,accuracy and durability,and analyzed for the applicability,according to the related requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).Results All the original cells without reporter gene transfection were negative,while all the four reporter cell lines were positive,and the negative and positive regions in dPCR results were clearly distinguished;The relative standard deviation(RSD) of the eight repeated detections of the same genomic DNA sample and the six independent extractions of genomic DNA sample of the same cell were all less than 10%,and the linear fitting R~2 values were more than 0.99 for both Luc and RPP30.The spike recoveries of five groups of samples detected by the developed method were between 50% and 100%,and the results of chip-type dPCR and droplet-type dPCR were consistent.This method distinguished the relative copy number of Luc in different cell clones,and the results of detecting the relative copy number of Luc in three passages(P8,P12 and P31) were highly consistent.Conclusion The developed duplex dPCR method has good specificity,precision,linearity,accuracy,durability and applicability,and might be used to evaluate the stability of Luc gene in Luc2P reporter cell lines.
ABSTRACT
Objective To establish a novel method to screen for anti-varicella-zoster virus (VZV) compounds with our previously generated reporter cell line for VZV, MV9G. MethodsMV9G cells were directly infected with cell-free virus of Oka vaccine strain (vOka) for 2 hours( CFV direct-infection) or cocultured with vOka-infected MeWo cells containing cell-associated virus for 48 hours (CAV co-culture) to promote expression of the reporter gene firefly luciferase. Antiviral compounds including heparin, mannose-6-phosphate( M-6-P), acyclovir( ACV ), resveratrol and roscovitine were added in the medium before or after the virus infection. Inhibitory effects( IC50 ) of the antiviral compounds were analyzed by comparing firefly luciferase activities of MV9G cells in the presence of antiviral compounds with those in the absence. Results Antiviral compounds inhibited luciferase activities of MV9G cells activated by CFV direct-infection and/or CAV co-culture in different levels. The reductions of luciferase activities statistically correlated with those of viral foci shown by immunostaining with a monoclonal antibody against VZV immediate early 62 antigens (IE62) in controls. Among these compounds, heparin, M-6-P, and 2.5 μmol/L of roscovitine inhibited CFV-activated more strongly than CAV-activated luciferase activities, whereas ACV and resveratrol inhibited CAV-activated more strongly than CFV-activated luciferase activities. Cell-associated ACV-resistant strains,Kanno and rOka YSR, activated luciferase activities of MV9G cells, too. However, the inhibitory concentrations (IC50) of ACV to the ACV-resistant strains were much higher than those to the ACV-sensitive strains,pOka and CaGu. ConclusionThe CFV direct-infection and CAV co-culture assays were useful to screen for antiviral compounds targeting the early and late phases of VZV infection, respectively. The VZV reporter cell-based assays may provide a simple, rapid, sensitive, and high-throughput method to screen for anti-VZV compounds.
ABSTRACT
Objective To establish the reporter cell lines for varicella-zoster virus(VZV)with ORF9G,the shortest and efficient sequence of the promoter for VZV ORF9,and ORF61F,the shortest and efficient sequence of the promoter for ORF61,and to characterize the cell lines.Methods The tandem promoters.T9G and T6lF,which were resulted respectively from the linkage of ORF9 in duplicate and of ORF6lF in duplicate.were cloned respectively into an individual reporter plasmid pGL3-basic.In this way,two recombinant promoter-reporter plasmids.pGL-T9G and pGL-T61F were constructed,in which the expression of reporter gene firefly luciferase was under the control of the upstream T9G or T61F.Along with the G418-resistant plasmid pCMV-script.the pGL-T9G and pGL-T6lF were respectively transformed into an in dividual Me Wo cell line.The grown G418-resistant cell clones were collected,and their firefly luciferase expressions post VZV infection was assayed.The best cell clones that have high firefly luciferase activity were chosen as reporter cell lines for VZV,of which the sensitivity and specificity were characterized. Results The activity of T9G or T61 F was two-fold as that of 9G or 61F.Two reporter cell lines,MV9G containing ORF9 ptomoter and MV6lF containing ORF61 promoter,were established successfully.Both cell lines showed fast.sensitive and specific response to VZV infection in a dose-dependent manner although the sensitivitv of MV9G Was somewhat higher than that of MV61F.Conclusion Each of both reporter cell lines for VZV may serve as a sensitive and specific research tool for further study especially on virus entry and antivi ral mechanisms.