Construction Of MiR-134Eukaryotic Expression Plasmid Vector And Lentivirus Vector And Verification Of Their Preliminary Validation

Background and Objective：Depression is a class of common diseases and the patients arepessimistic about the future, have no interest in daily activities andentertainment, tend to suffer from anxiety and sleep poorly. So far, theetiology and pathogenesis of depression were rarely clear. But what is sureis that biological, psychological and social environment factors involved inthe pathogenesis of depression. In recent years, certain studies suggestedthat mental illness was associated with miRNAs, which may play a veryimportant role in the occurrence and development of mental illness.Studies have found that the expression level of miR-134wassignificantly reduced in patients with bipolar disorder manic plasma,however, its expression was obviously elevated in the rat model of statusepilepticus and human temporal lobe epilepsy patients. The current studyconfirmed that the abnormal expression of miR-134was related to mentalillness, but the study of mental illness in the fine regulation of miR-134was still very scarce. To further explore the function of miR-134, we need to build the cell or animal models and detect its changes in thephysiological and pathological aspects in vitro and in vivo.This topic aims to build miR-134eukaryotic expression vectorplasmid and lentivirus vectors, and transfect them into OL cells and SD rathippocampal neurons to provide an effective tool for the subsequent studyof molecular mechanisms of depression.Methods：1. The fragment containing the gene of miR-134from OLcell genomewas amplificated by PCR, and then the PCR product and the vectorpEGFP-N1were ligated to construct a vector pEGFP-miR-134.Therecombinant plasmid was transiently transfected into OL cells usingInvitrogen′Lipofectamine LTX and Plus Reagents plasmid transfection kitafter digestion and gene sequencing. The total RNA was extracted and theexpression of miR-134in OL cells was detected in the genetic level.2. The hippocampal neurons of clean grade SD rats born within24hwere cultured using the serum free medium to lay a foundation for theexperiment of cell vitality, cell apoptosis and synaptic plasticity inhippocampal neurons.3. The Lenti-miR134-EX lentiviral vector was constructed and thelentivirus was packaged and purified. Then, the hippocampal neurons ofSDrats were infected with identified lentivirus. The total RNA wasextracted and the expression of miR-134in the hippocampal neurons was detected in the genetic level.Results：1. The fragment containing the miR-134gene was successfullyamplified from OL cell genome and successfully connected into the vectorpEGFP-N1. The recombinant plasmid was named pEGFP-miR-134.Thetransfection effect was better.2. The high purity SD rat primary hippocampal neurons were isolatedand cultured using the simple method. The purity of hippocampal neuronswas more than90%using MAP-2immunocytochemistry method afterseparating at least7days.3. The lentiviral vector expressing green fluorescent protein, namedLenti-miR134-EX, was successfully packaged.Conclusion：This research successfully established the eukaryotic expressionvector plasmid and lentivirus vectors, got the transiently transfected humanglioma cells and primary hippocampal neurons of SD rats, and providedeffective tools and platforms for further research of miR-134in depression.