The Construction And Molecular Imaging Of RNA-reporter Systems In Living Subjects

RNA plays an important role in many biological processes such as the role in regulation of gene expression.It requires RNA processing to become a messenger RNA(mRNA)during RNA maturation in eukaryotes from which RNA transcription is spliced.Many different types of RNA show tissue specific pattern and level of expression in various physiological or pathological conditions.For example,the amounts and kinds of miRNA show great differences.Both the post-transcriptional RNA processing and the tissue specific expression of miRNA play a very important role in physiological process.Therefore,it needs to detect RNA content to study the biological activities of RNA.The expression levels of RNA are measured using the conventional biochemical approaches in vitro such as RT-PCR,Northern blotting,RT-qPCR,microarray assay and RNASeq.However,these methods require destruction of cells and tissue samples,thus it is impossible to provide dynamically information of rapid fluctuation on RNA contents which is important to accurately study the relevant biological processes.The reporter gene is an important tool for RNA detection because it can be assembled and expressed in vivo to produce detectable signal molecules.It can respond to the changes of relevant RNA and detect RNA content in real time.At the same time,reporter gene can more truly simulate the biological and biochemical reactions and more truly simulate the processing of RNA,and thus the detection results are more close to the real physiological process of body.In this study,we developed three bioluminescence reporter systems to dynamically monitor the RNA changes which are useful tools for RNA detection.1.Construction of an intron retained bioluminescence reporter and its in vivo imaging study.Pre-mRNA splicing is a key step in the information transfer between DNA and proteins in eukaryotes.Due to the lack of non-invasive methods for real-time monitoring of pre-mRNA splicing events in vivo,in this chapter,an intron retained reporter gene,Rluc-intron,was developed to noninvasively monitor the splicing process in living subjects.We inserted the renilla luciferase(Rluc)gene into the intron sequence of the human TPI(human triose phosphate isomerase)gene.The splicing process was simulated and dynamically monitored using the reporter gene regulated by splicing inhibitor in vitro and in vivo.With these experiments such as the activity verification of reporter gene and the response of reporter gene to splicing inhibitors etc,we proved that the Rluc-intron imaging2.system could respond to the modulators on pre-mRNA splicing and could provide dynamic information of the pre-mRNA splicing in living subjects.3.A two-step transcriptional amplification method for pre-mRNA splicing imaging in living subject.In the previous chapter,the Rluc-intron reporter gene for detecting the pre-mRNA splicing was constructed by inserting the Rluc gene into the intron sequence.However,when the intron sequence is almost completely removed,in this case,it is difficult to detect the bioluminescent signal resulted from Rluc.Therefore,the system can hardly reflect the weak activity of splicing modulator on splicing regulation.In order to overcome this defect,a pre-mRNA splicing associated gal4-vp16 imaging system was designed in this chapter for dynamic study of pre-mRNA splicing process by using the activation of gal4-vp16 to activate UAS(upstream activating sequence)and then enhanced the downstream firefly luciferase(Fluc)gene expression.By detecting dynamic changed bioluminescent signals in living subjects,We demonstrated that the reporter gene system could well respond to the splicing regulator and the pre-mRNA splicing process was well monitored.This will effectively expand the application of intron retained reporter gene system for real-time and dynamic monitoring of pre-mRNA splicing.4.Design of a dual luciferase reporter gene system and its application in the detection of miRNA in neural differentiation process.Monitoring the neural differentiation is crucial to the study of neuronal development and stem cell therapy for neurodegenerative diseases.In this chapter,we developed a dual luciferase reporter system for monitoring the expression changes of neuron-specific miR-9 and miR-124 a in vivo and in vitro.The miRNA responsed reporter gene was constructed by inserting Fluc and Rluc into the same vector and initiated by two different promoters.At the same time,three completely repeat sequences complemented to miR-9 or miR-124 a were inserted into the 3' non-coding region of Fluc to response to the corresponding miRNA expression.Rluc is not regulated by miRNA and can be expressed independently.By transfection with exogenous miRNAs,we demonstrated that the dual luciferase reporter system could effectively and specifically detect the miRNA changes.Using this reporter gene,we successfully detect the miR-9 and miR-124 a during neural differentiation.The dual luciferase reporter system provides a powerful tool for continuous monitoring of miRNA expression in living subjects.