The Construction Of Recombinant Adenoassociated Virus Vector Encoding Cdk5-siRNA And The Role Of CDK In The Neurodegeneration Of NPC

ObjectiveInvestigate the impairment of neuronal cytoskeleton caused by activation of cdks in different aged npc mice ,in order to study the pathogenesis and explore the new therapies for NPC.MethodsSacrifice all sibling mice in one breeding cage at the age of 1,2,3,4 and 5 week, keep the npc-/- mice and same aged wild type mice as control (at least 4 mice per group). Use PCR for genotyping and immunohistochemistry for the study of spheroid caused by activation of cdk.ResultsAxonal spheroids caused by activation of cdk can be found as early as 3 weeks age in npc mice.The pathology is more obvious in the cerebellum compared to pons.The number of spheroids increased as the mice grow up.Conclusion①The impairment of neuronal cytoskeleton and cdk activation can be found in npc mice as early as 3 weeks age.②The severity of pathology increased along with the increase of age. ObjectiveTo construct the adeno-associated virus vector encoding the small interfering RNA(siRNA) specific for cyclin dependent kinase5(CDK5) gene and to observe its silencing effect on cdk5 gene in vitro,in order to explore the role of the CDK5 in the neurodegenerative disease.MethodsThe specific cdk5-siRNA sequence was cloned into the plasmid of pAAV-MCS-EGFP in AAV Helper-Free System to construct the rAAV2 expression plasmid pAAV-MCS-EGFP-U6-cdk5-siRNA. The recombinant plasmids were identified by DNA sequencing and restriction digestion. Then the packaging cell line (HEK293 cell) was co-transfected with the pAAV-MCS-cdk5-siRNA together with the control plasmid pAAV-RC and pHelper by phosphate-calcium deposit method. The titer was measured by dot hybridization. Infected the PC12 cell using recombinant adeno-associated virus, at 72h after infection, the expression of cdk5 was detected by Western-blot.ResultsAll plasmids and the recombinant adeno-associated virus vector carrying the cdk5-siRNA sequences were constructed successfully, the viral titer was 4×1013,it can infect the PC12 cell and down-regulated the expression of the cdk5 gene in PC12 cell.ConclusionThe recombinant adeno-associated virus vector rAAV-cdk5-siRNA can interfere with the expression of cdk5 gene significantly, which lays the basis for its application in the treatment of the neurodegenerative disease.