Construction of small interfering RNAs against mouse nuclear transcription factor Rbpj by recombinant adenovious and its inhibitory during osteogenetics induced by BMP9 in C3H10T1/2

Abstract:

Objective：To construct recombinant adenovirus against mouse nuclear transcription factor Rbpj，downstream of Notch，with the purpose to provide a powerful molecular tool for exploring the role of bone morphogenetic protein（BMP） ９ signaling in osteogene－sis of mesenchymal stem cells. Methods：Three pairs of double-stranded DNA sequence targeted mouse Rbpj were designed and sub－cloned to pSES-HUS vector to obtain pSES-HUS-siRbpj using AdEasy system，then reorganized with pAdEasy-1 plasmid in E.coli BJ5183. pAd-siRbpj was transfected into HEK293 to package recombinant adenovirus AdsiRbpj after being linearized by PacⅠand screened by RT-PCR and Western blot. C3H10T1/2 were co-treated with recombinant adenovirus and BMP9 proteins，then the early osteogenetic maker alkaline phosphatase（ALP） was detected at the 7th d. Osteopontin（OPN） and osteocalcin（OCN），BMP downstream target genes Id 1 and Runx 2 expression were also detected by RT-PCR. Results：Three interference recombinant adenoviruses were successfully constructed，of which AdsiRbpj-2 had the best interference ability and can significantly decrease BMP9-induced os－teogenic indexes，including the early osteogenetic index ALP，late osteoblasic differention indicators OPN，OCN and osteogenic gene Runx 2. Conclusions：The siRNA adenovious vector AdsiRbpj-2，with the ability to specifically block mouse Notch signaling，is suc－cessfully constructed，future indicating that Notch signaling pathway plays an important role in osteogenic differention of BMP9 induced bone marrow mesenchymal stem cells.