title: [Overexpression of mouse IRF8 inhibits the differentiation of RAW264.7 cells into osteoclast-like cells].
abstract: OBJECTIVE
  To construct and identify recombinant adenovirus Ad-IRF8 carrying mouse interferon regulatory factor-8 (IRF8) and observe the influence of the over-expressed IRF8 on the differentiation of osteoclast precursor cells (RAW264.7) induced by soluble receptor activator of nuclear factor κB ligand (sRANKL).
  
  
  METHODS
  Mouse IRF8 gene amplified by PCR was subcloned into the shuttle vector pAdTrack-CMV. The vector pAdTrack-IRF8-CMV was linearized by Pme I , followed by homologous recombination with bone vector pAdEasy-1 in E.coli BJ5183. Recombinant adenovirus vector pAd-IRF8 was obtained and linearized by Pac I , and then transfected into AD293 cells. The recombinant adenovirus Ad-IRF8 was harvested after packaged in AD293 cells. RAW264.7 cells were infected by Ad-IRF8. The overexpression of IRF8 was verified with semi-quantitative reverse transcription-PCR (RT-PCR) and Western blotting. The influence of the overexpression of IRF8 on sRANKL-induced differentiation of RAW264.7 cells was detected by tartrate-resistant acid phosphatase (TRAP) staining.
  
  
  RESULTS
  The shuttle vector pAdTrack-IRF8-CMV and recombinant vector pAd-IRF8 were successfully constructed. The recombinant adenovirus Ad-IRF8 was also successfully packaged in AD293 cells. The overexpression of IRF8 in RAW264.7 cells was confirmed by RT-PCR and Western blotting. TRAP staining showed that the overexpression of IRF8 effectively inhibited the differentiation of RAW264.7 cells into osteoclast-like cells.
  
  
  CONCLUSIONS
  The recombinant adenovirus Ad-IRF8 was successfully constructed and the overexpression of IRF8 was also detected in RAW264.7 cells. The overexpressed IRF8 can effectively inhibit the differentiation of RAW264.7 cells into osteoclast-like cells.
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Wenlong
authors.initials: W
authors.lastname: Yan
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Hai
authors.initials: H
authors.lastname: Yao
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Tingmei
authors.initials: T
authors.lastname: Chen
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D059765:Homologous Recombination; D050835:Interferon Regulatory Factors / Q000235:genetics*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D000099185:Interferon Regulatory Factor-8
chemical_list: D050835:Interferon Regulatory Factors; D000099185:Interferon Regulatory Factor-8