| Objective: To construct the CRMP-1-phrGFPâ…¡-N mammalian expression vector and test the effects of up-regulation of the CRMP-1(Collapsin Response Mediator Protein-1) gene on axon outgrowth.Methods: To extract the total RNA from the hippocampus tissue of a 6 days SD rats and amplify the cDNA of CRMP-1 gene by RT-PCR technique, the products were cloned into PGEM-T Easy, sequenced and then subcloned into mammalian expression vector phrGFPâ…¡-N. The recombined vector was then transiently transfected into PC12 cells stimulated by NGF to differentiate into neuron-like cells, observed the subcellular localization of CRMP-1 protein and changes in phenotype of PC12 cells by fluorescence microscope.Results: 1) Successfully amplified the cDNA of CRMP-1 gene, and constructed the CRMP-1-phrGFPâ…¡-N mammalian expression vector with base pairs identity is 99.8% homologous to the sequence acquired from genebank. 2) Neurites of PC12 cells which were not induced by NGF were shot and had few branches, proliferated slowly. Neurites of the PC12 cells which induced by NGF extended evidently and branched greatly. After transfection, the cells transfected into phrGFPâ…¡-N shew green fluorescence both in cell body and in neurites, but the fluorescence in the end of neurites and cell body is stronger than that in the neurites. The length and branches of this group of cells are similar to un-interfered control group. Cells transfected into CRMP-1-phrGFPâ…¡-N show weak green fluorescence but their distribution resembles the cells transfected into phrGFPâ…¡-N. But it's obviously that this group of cells displays short neurites and fewer branches.Conclusions: 1) The CRMP-1-phrGFPâ…¡-N mammalian expression vector can be constructed successfully. 2) CRMP-1 Protein could express both in cell body and in neurites of PC12 cells. 3) Short neurites and fewer branches indicate that excessive expression of CRMP-1 protein probably inhibits the outgrowth and branching of neurites. |
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