Construction Of NeuroD Lentivirus Expression Vectors And Over-expression In Rat Neural Stem Cells

Background and Objective: In mammals,NeuroD1 is widely expressed during brain development and isessential for the development of the central nervous sytem,particularly for the generation of granule cells in the hippocampusand cerebellum.NeuroD1-expressing cells are not radial glial orprogenitors cells. NeuroD1 gene is up-regulated in new born cells duringpost-metamorphic neurogenesis. To construct the lentivirus vectors carrying NeuroD genes,and verify their expression in neural stem cells(NSCs).Methods: A pair of specific primers was designed on the basis of cds region of rat NeuroD and the DNA gene was amplified by PCR, with rat brain cDNA serving as template. The PCR products was obtained by gel purification. After identified by the ligation reaction, the target fragments were cloned into lentivirus vectors,which was recombined in DH5 a bacteria. The recombinant pHB-NeuroD lentivirus particles were produced by transfection into 293 cells.The neural stem cells were derived from the brain tissue of rat embryo at the age of 13-15 days and proliferated steadily under the serum-free medium which contained B27,basie fibroblast growth factor(bFGF)andepidermat growth factor(EGF).the rat neural stem cells were identified by Nestin inamunofluorcscence. recombinant pHB-NeuroD lentivirus particles subsequently infected NSCs. The NeuroD mRNA levels were detected by QPCR.Result: in this experiment, the lentivirus vectors were proven successfully constructed with high infection efficiency,and the 13-15 days pregnant SD rat fetal rat of NSCs were isolated and identified in vitro. and NeuroD was stably over-expressed in rat NSCs.Conclusion: The recombinant pHB-NeuroD was successfully constructed,which provided a basis for futher study of the effect of NeuroD on NSCs.