Construction And Identification Of RNAi Lentivirus Vector Targeting Rat RAG1 Gene

Recombination activating genes(RAGs)were a group of stable DNA fragments which had the ability to activate recombination proceeding,discovered by Schatz in the1980s last century in immune system.They had two subtypes,RAG1 and RAG2. RAGs played a critical role in the development and maturation of lymphocytes,and were related to immune memory function.It had been proved that knockout of RAG1 could lead to defects of immune memory.With further reseach,people found that RAGs did not only exit in immune system,but in nervous system of mammal, amphibian animals and Osteichthyes.Chun detected the low levels of RAG1 transcript in the murine central nervous system by polymerase chain reaction,in situ hybridization and Northern Blot analyses.The RAG1 transcripts were found to be widespread in embryonic and postnatal neurons,with transcription being most apparent in regions of the postnatal brain with a high neuronal cell density---the cerebellum and the hippocampal formation,both of which were relevant to learning and memory function.Thus,that researching whether RAG1 expressed in brain regions relevant to learning and memory was concerned with learning and memory, had an important significance.Although knockout of RAG1 could inhibit its effects on immune memory,it might contribute to dysplasia of brain during embryonic stage, and,accordingly,affect function of postnatal brain.So it was apparently unfeasible to utilize the technology to research the learning and memory function of RAG1 in nervous system. RNA interference(RNAi)was a new technology used to study the function of interest genes.Compared with gene knockout method,it had so many advantages, such as make interest genes get real time inhibition,almost no impacts on the organic development of animals,much more suitable to study the function of interest genes,and making operation more simple and convenient.RNAi technology in the earlier period had a lower transfection efficiency in vivo due to being affected by vectors,and it was only used in vitro,and not in vivo.With the further research to virus vectors,RNAi segments could be packaged by virus vectors and introduced into vivo,and transfection efficiency had a dramatic enhancement making application range of the technology from vitro to vivo.However,we hadn't seen the documents reported about using RNAi to study the function of RAG1.To implement total goal of the research about the role of RAG1 in brain function, this topic constructed lentivirus expression vector carrying RAG1 interference segments.The research will lay the foundation for the follows:making the study of RAG1 functions in vivo come true and illustrating the relations between RAG1 and learning and memory function.Objective:To construct RNAi lentivirus expression vectors and observe the transfection efficiency on cultured rat hippocampus neurons and inhibitory effects on RAG1, respectively,and screen one which can block the expression of RAG1 high effectively and specificly.Methods:1.Construction of RNAi recombinant lentivirus vectors mediati -ng RAG1 gene silencingThree pairs of shRNA sequences targeting RAG1 were designed,named shRNA1,shRNA2,shRNA3,and pRNAT-U6.2/Lenti vectors were digested by restriction endonuclease BamHâ… and Xhoâ… .The three segments of shRNA were ligated into pRNAT-U6.2/Lenti vectors,respectively,and the products pRNAT-U6.2/ Lenti-shRNA1/RAG1,pRNAT-U6.2/Lenti-shRNA2/RAG1,pRNAT-U6.2/Lenti-shR -NA3/RAG1 were identified and selected,pRsv-Rev,pMDLg/pRRE,pMD2.G and pRNAT-U6.2/Lenti-shRNA1/RAG1,or pRNAT-U6.2/Lenti-shRNA2/RAG1,or pRN -AT-U6.2/Lenti-shRNA3/RAG1 were cotransformed into the 293T cells by lentivirus vector system,respectively.Cultured cells in vitro were infected by lentivirus and their infection efficiency was observed.2.RT-PCR and real-time fluorescence quota PCRHarvest hippocampus neurons after being infected 96 hours,extract total RNA, and detect the expression levels of RAG1 mRNA by RT-PCR and real-time fluorescence quota PCR.RT-PCR products were analyzed by electrophoresis.After real-time fluorescence quota PCR,we performed dissociation curve analysis to RNA products and checked if there was any bimodal dissociation curve or abnormal amplification plot,then,we obtained its fluorescence cycle threshold value from fluorescence quota PCR dynamics curve of each group of samples,compared with the standard curve and calculated RAG1 copy number of each unmeasured group to examined the expression level of RAG1mRNA.3.Western blottingHarvest the hippocampus cells after being infected 96 hours and extract proteins. Then,they were taken to PAGE,and transferred to PVDF membrane and incubated with mouse-anti-RAG1 antibody,horseradish peroxidase marked goat-anti-mouse antibody,respectively.We observed results by ECL.Results:1.The positive recombination pRNAT-U6.2/Lenti-shRNA1/RAG1,pRNAT-U -6.2/Lenti-shRNA2/RAG1 and pRNAT-U6.2/Lenti-shRNA3/RAG1 were obtained after shRNA1,shRNA2,shRNA3 was ligated into pRNAT-U6.2/Lenti,and the shRNA1,shRNA2,shRNA3 could be successfully transduced into pRNAT-U6.2/ Lenti by verification.Recombination lentivirus vectors in high titer were obtained by lentivirus vector system and could be transformed effectively into cultured cells in vitro,making them produce siRNA. 2.An obvious reduction of the expression of RAG1 mRNA and protein in cultured hippocampus neurons after transfection with lentivirus expression vectors compared with that of control vector-treated and untransfected hippocampus neurons (P<0.05).The pRNAT-U6.2/Lenti-shRNA3/RAG1 was the one that had the strongest inhibitory effect,and inhibitory ratio to protein and mRNA was(67+5.8)%and (77+5.0%).But RAG1 mRNA and protein expression levels were unaffected in control vector-treated and untransfected cells(P>0.05).Conclusion:RNAi lentivirus expression vectors have been established and screened first sucessfully,which can block the expression of RAG1 high effectively and specificly, and there titers are relatively higher.They offer a new method to further research the expression of RAG1 that can be inhibited in vivo and to study the function of RAG1 protein in central nervous system.