关键词: RNA干扰, shRNA, 腺病毒载体, 悬浮细胞

Abstract: Abstrac: Objective Constructed recombinant adenovirus to efficiently obtain gene silencing in suspension cell. Methods Inserted 76 nt DNA stem-loops into plasmid pRNAT-H1.1/Adeno, in which an incorporated GFP reporter allows convenient tracing of all steps in viral production. In this pilot study, placental growth factor (PLGF) was targeted by this vector. The recombinant shuttle vector were linearized with PmeⅠ and treated with alkaline phosphatase, then cotransformed with pAdeasy-1 into BJ5183 cells by electroporation. Positive clones were selected and confirmed by PacⅠdigestion. Before transfected the packaging cell line HEK 293, the recombinant adenovirus plasmid must be cut by PacⅠ. After three times amplified in HEK293 cells, high titter viral particles were harvested and quantified by OD260 optical absorbance and TCID50 method individually. Finally, the viral particles were utilized to transfect two suspension cells (NCI-H 250 and NCI-H 209, the human small cell lung cancer cells). And using real-time PCR to test the target gene expression and Transwell cancer cell migrate test to analysis cancer cells’ function. Results Monitoring GFP expression and using real-time PCR method, efficient and specific silencing of PLGF gene was found in NCI-H 250 and NCI-H 209 cells after adenovirus transfection 48 h. And a significant reduction of migration was observed in target gene silenced cancer cells. Conclusion Our results indicate a prospective application of this shRNA expressing recombinant adenovirus system in those cells transfected difficultly by other methods.

Key words: RNA interferenc, shRNA, adenovirus, suspension cell