| Objective:To construct the CRMP-5-pEGFP-C2 eukaryotic expression vector and investigate the positive effect of up-regulation of the CRMP-5 gene on neurite outgrowth of hippocampus neurons in rats.Materials and methods:To extract the total RNA from the hippocampus tissue of a 6 days SD rats and amplify the cDNA of CRMP-5 gene by RT-PCR technique,the products were cloned into pEGFP-C2 eukaryotic expression vector.The recombined vector was then transiently transfected into hipppcamups neurons,at last,to observe the localization of CRMP-5 protein and changes in neurite by fluorescence microscope.Results:(1) The cDNA of CRMP-5 gene were amplified successfully,and the CRMP-5 -pEGFP-C2 eukaryotic expression vector was constructed,the basal pairs of which were absolutely homologous with the sequence acquired from genebank.(2) After transfection,the fewer cells transfected by pEGFP-C2 vector showed green fluorescence both in cell body and neurites evenly,the length and branches of this group of cells were similar to un-interfered control group.On the contrary,more cells revealed green fluorescence which was stronger in the end of neurites and cell body than that in the middle of neurites since CRMP-5-pEGFP-C2 vector was transfected into.Meanwhile,the neurites of cells were obviously longer than which of cells in un-interfered control group and pEGFP-C2 vector group.Length of neurites demon-strated a tendency to be gradually extended accompanying expression of CRMP-5 protein and neurites formed sparse network at last.Conclusions:(1) The CRMP-5-pEGFP-C2 eukaryotic expression vector is constructed successfully by cloning technology.(2) CRMP-5 protein is expressed both in cell body and neurites of hipppcamups neurons.(3) Long neurites and numerous branches of cells which overexpress CRMP-5 protein suggest that CRMP-5 probably play a important role in promoting neurite outgrowth and branch in hipppcamups neurons.
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