Rational Design Of An Activatable Reporter Quantitative Imaging Of RNA Aberrant Splicing In Vivo

Objective: Studies have shown that the mature processing of RNA is an important part of gene expression.Pre-m RNA splicing is an important stage of gene expression in most eukaryotes.RNA splicing events are also key to many diseases,especially the pathology of cancer.In order to achieve the treatment of tumor diseases,it is an effective and feasible therapeutic strategy by using pre-m RNA splicing to specifically interfere with gene expression at the molecular level.In recent years,the expression of genes at the m RNA level has been restricted by traditional biochemical methods.These methods can usually only be detected in vitro,and can not avoid molecular-specific effects,nor can they accurately monitor pre-m RNA splicing in real time,which hinders pre-m RNA splicing imaging research in vivo.Therefore,it is very necessary to develop ideal molecular probes for visualizing pre-m RNA splicing in vivo and high-throughput screening of splicing regulators,which will benefit the treatment of human diseases induced by pre-m RNA abnormal splicing.Methods: In this study,we designed an activatable bioluminescence reporter gene for imaging pre-m RNA abnormal splicing in vivo.The reporter gene Rluc-SMN was designed by inserting a small human survival motor neuron 2(SMN2)gene upstream of Renilla luciferase(Rluc),so that only unspliced m RNA can translate active luciferase protein.After that,we used the method of luciferase reporter system to detect the response of luciferase activity to the drug dose and drug action time under the stimulation of foreign drug flavonoids(ISO),and to make RT-PCR analysis.Finally,bioluminescence imaging technology was used to monitor abnormal splicing of pre-m RNA in animals.Results: After transfecting the reporter gene Rluc-SMN constructed by us,the measured luciferase intensity is significantly lower than that of Rluc-control and Rluc-mutant,which indicates that the splicing reporter gene we constructed can be used for pre-Monitoring of m RNA splicing.Rluc-SMN or Rluc-mutant constructs of different concentrations(0,0.25,0.5,1,2 ?g / ml)were then transfected into 293 cells.CKK-8 assay results showed that splicing reporter gene had no effect on cell viability.First,complete the treatment of foreign drug flavonoids(ISO)at the cellular level.It was detected that with the increase of drug concentration and the increase of drug treatment time,the expression level of luciferase increased,showing drug dose and time dependence,indicating that the constructed The splicing reporter gene can sensitively monitor splicing.Secondly,the results of RT-PCR analysis and luciferase detection have a consistent trend.Finally,the luciferase reporter gene we constructed can be imaged in vivo in small animals.Through the analysis and observation of the imaging results,it can be concluded that the splicing process is inhibited after treatment with splicing inhibitors.In conclusion,the successful construction of the luciferase reporter gene provides a new means for real-time monitoring of abnormal splicing of pre-m RNA in vivo.Conclusion: The luciferase reporter gene Rluc-SMN developed in this study can effectively monitor abnormal pre-m RNA splicing in vivo in real time.In principle,this reporter gene can potentially be used for non-invasive visualization of splicing-dependent processes and high-throughput screening of splicing regulators in cells.