Encoding Rat Rgma Shrna Eukaryotic Expression Vector And Its Interference With The Efficient Screening Identified

Objective: To establish oxygen-glucose deprivation axon model of rats'hippocampal neurons in vitro. To use this model as a trail base for research involve the injury of oxygen glucose deprivation and axons regeneration.Methods: The hippocampal neurons cultured for 7 days were exposed to D-hanks solution with nitrogen gas in stead of the original culture medium for 0.5～1.5h, then continue to be cultured in the original culture medium with oxygen. Testing the level of LDH at different time in culture medium and observe the change of axon.Results: The refraction of hippocampal neurons is decreasing and the swelling of cells is obvious, the length of the axon is shorter than before with the number of LDH is increasing. Besides, the survival ratio of hippocampal is higher and the change of axon length is more obvious in the group of oxygen-glucose deprivation 0.5h than in the other groups.Conclusion: Successfully established oxygen-glucose deprivation axon model of rats'hippocampal neurons in vitro. Objective: To construct three plasmids with RGMa shRNA for interfering RGMa and selected one sequence RGMa shRNA with the highest gene silence effectiveness for the next trial.Method: Searching the mRNA sequence of RGMa from the website of pubmed and designing threes strips shRNA to silence the mRNA of RGMa. Then, take the target shRNA into plasmid of pegensil-1. Increasing the number of pegensil-1 includes 1, 2 and 3 with shRNA and without endotoxin. Culturing the cell of PC12 and using the plasmid with shRNA and without endotoxin to transfect PC12 cell. There are four groups, one is the transfected by the negative control plasmid, and the last three groups are transfected by the 1, 2 and 3 with shRNA. Testing the effectiveness of gene silence in 48h by RT-PCR and Western blotting. Results: one plasmid of 1, 2 and 3 with shRNA is the most effective to interfere the mRNA of RGMa.Conclusion: successfully constructed the plasmid with shRNA and selected the one plasmid with highest gene silence effectiveness...