Role Of Fyn In The Process Of Neuronal Migration

In order to study the role of Fyn in neuronal migration during brain development, RNAextracted from mouse brain tissue was taken as a template, and mouse Fyn gene was clonedusing PCR, eukaryotic expression vector were constructed. Fyn shRNA sequences andnegative control sequence were designed depending on the mouse Fyn mRNA sequence andthe online design tool and interference vectors were constructed. Cells were transfected bythese vectors, the expression of Fyn was detected in RNA and protein level, effectiveness ofthe vectors was identified and the effective interference sequence was chosen out, then inutero electroporation experiment was carried using the recombinant vectors to discusse therole of Fyn in neuronal migration. The results were as follows:1. Mouse Fyn gene containing CDS was cloned succesfully and eukaryotic expressionvector pM-Fyn was constructed. The results showed that the recombinant vector did not havemutation point and deletion. Chinese hamster ovary (CHO) cells were transfected withpM-fyn and the expression of Fyn was detected. Results showed that the Fyn expression wassignificantly increased in RNA and protein levels in the cells which were transfected with therecombinant vector;24h after transfection, cells were immunofluorescence staining, and theoverexpression of Fyn caused cells produced filopodia.2. Results showed that the shRNA sequences were same as design and the interferencevectors were successfully constructed.293T cells were transfected in order to choose theeffcective sequence. The expression of Fyn was detected in RNA and protein level,compaired to conrrol group, shFyn-1could decrease the expression of Fyn.3. In utero electroporation experiments were carried using the overexpression andinterference vectors. Results showed that compared to the negative control, neuronsoverexpressed Fyn had more branches during migration, and neurons had longer leadingprocess when the expression of Fyn was decreased, but did not affect the formation of thenormal layered structure of the cerebral cortex, neurons could migrate to the correct position.The results showed that, overexpression of Fyn induced filopodia generation in culturedCHO cells, and migrating neurons had more branches, suggesting that Fyn may change the cell shape by affecting cytoskeletal rearrangements. The migrating neurons inhibited theexpression of Fyn had longer leading process in the final stages of the migration, indicatingFyn may play an important role in somal translocation.