CRMP4 Gene Knockout Effects On Dendritic Spine Formation In Neurons

Objective:To observe the variation of dendritic spine formation in rat hippocampal neurons by CRMP4 gene knockout. It is aim to explore the effect of CRMP4 gene in dendritic spine.Methods:Firstly, The si RNA fragment was designed & ordered. Secondly, 293 T cells were cultured. Afterwards, the recombinant plasmid of CRMP4 and si RNA or negative fragment should be transfect respectively in 293 T cells with lipofection reagent. In order to verify the si RNA fragment interference efficiency, it is require to extract cell lysate’s protein, which was used to do Western Blot experiment and detected by the internal antibody GAPDH at the same time. At last, SD rat hippocampal neurons were cultured in vitro. Calcium and Phosphorus need to be transfect when rat hippocampal neurons were cultured three days later. The purpose is to knockout CRMP4. Meanwhile, we adopt immunofluorescent staining combine with laser confocal microscope method to observe and analyze dendritic spines formation in system.Results:(1)The si RNAs and negative fragment were transiently transfected into 293 T cells with lipofection reagent. The cells grow well and intracellular distribute green fluorescent;(2)The Western Blot experiment results showed that CRMP4 content is less,almost hardly express. There is no significant difference between the two si NRA groups. However, negative controlled group content is more, but the electrophoresis bands of internal antibody GAPDH has no obvious difference in the three groups;(3)Hippocampal neurons were cultured in vitro. The cell inoculating density is about 1×105/ ml;(4)Primary cultured hippocampus neurons knock the CRMP4 gene out. There is a few neuritis hippocampal neurons and moreover dendritic surface green fluorescence expression is less. The expression of PSD95 which is marked red fluorescence is also declined. The two groups showed the same results either after fluorescence coincidence. Compared with the controlled group, it is exist statistics difference.Conclusions:(1)The candidate si RNAs were transiently transfected into 293 T cells with lipofection reagent;(2)The si RNA fragment interference efficiency is good;(3)Hippocampal neurons is successed to be cultured in vitro;(4)The density of dendritic spines decreased significantly after si RNA interference CRMP4. It is suggested that the CRMP4 gene knockout could inhibit the formation of dendritic spines in hippocampal neurons.