title: [Construction and identification of recombinant retroviral vector containing human interleukin 1 receptor antagonist and its expression in osteoarthritic human articular chondrocytes].
abstract: OBJECTIVE
  To construct the retroviral vector containing human interleukin 1 receptor antagonist (IL-1Ra) and to investigate the property of the transfected articular chondrocytes from osteoarthritic patients in vitro.
  
  
  METHODS
  Retroviral vector PLXRN carrying IL-1Ra (PLXRN-IL-1Ra) gene was constructed by inserting IL-1Ra gene at the sites of Sal I and BamH I. The recombinant retroviral plasmid was homologously recombinated in bacterial cells. After screening and amplification, the recombinant retroviral plasmid was obtained and transfected into PT67 cells. The replication-defective retrovirus PLXRN-IL-1Ra was packed and amplified in the PT67 cells. Viral titer was determined by infecting NIH/3T3 cells with serially diluted viral supernatants produced with a control vector. Experiments were divided into 3 groups: non-transduced group (group A), PLXRN transduction group (group B), PLXRN-IL-1Ra transduction group (group C). Primary articular chondrocytes from osteoarthritic patients were transduced with PLXRN and PLXRN-IL-1Ra. The positive chondrocytes clones, which were G418-resistant, were cultured for 3-4 weeks after being selected by G418. The expression of IL-1Ra mRNA in the chondrocytes was determined by RT-PCR. Levels of IL-1Ra protein synthesis in the supernatants were measured by ELISA.
  
  
  RESULTS
  Restrictive endonuclease identification and gene sequencing confirmed that the recombinant contained IL-1Ra cDNA. Virus titer could reach 3 x 10(4) CFU/mL. Primary chondrocytes cultured in vitro were polygonal or spindle and were stained with purple particles by toluidine blue staining. After stable transduction into the chondrocytes the 311 bp fragment of IL-1Ra was detected in group C by semi-quantitative RT-PCR. ELISA showed that IL-1Ra in supernatants of the group A and group B were below the level of detection. The concentrations were (60.47 +/- 15.13) ng/L in group C. There were significant differences between gene transduction group and control groups (P < 0.05).
  
  
  CONCLUSIONS
  The construction of recombinant retrovirus vector by homologous recombination in bacterial cells can be quickly and easily performed. Stable and effective expression of IL-1Ra can be achieved by transduction with retroviral vectors in osteoarthritic articular chondrocytes, indicating potential utility in gene therapy for osteoarthritis.
authors.affiliation: Department of Orthopedic Surgery, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200011, P.R. China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Rui
authors.forename: You
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Zhang
authors.forename: Hongli
authors.initials: H
authors.lastname: Sun
authors.forename: Zhihu
authors.initials: Z
authors.lastname: Qu
authors.forename: Kerong
authors.initials: K
authors.lastname: Dai
mesh_terms: D002358:Cartilage, Articular / Q000378:metabolism*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D053590:Interleukin 1 Receptor Antagonist Protein / Q000235:genetics*; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
chemical_list: D053590:Interleukin 1 Receptor Antagonist Protein