title: [Establishment of a transgenic cell line with stable expression of human CD14].
abstract: OBJECTIVE
  To establish a transgenic cell line with stable expression of CD14.
  
  
  METHODS
  Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR, T-A clone techniques and ABI PRISM377 machine. Eukaryotic expression vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonucleases EcoR I/Xba I and ligating with T4 ligase. The human cervical cancer cell line Hela was transfected with the positive recombinant plasmid pcDNA3.1(+)/CD14 using superfect transfection reagent. Positive clones were selected by G418 at a concentration of 0.5 μg/μl and the expression of human CD14 on the transfected Hela cells was confirmed by quantitative PCR and immunofluorescent assay.
  
  
  RESULTS
  There was significantly difference om expression of CD14 mRNA between the blank pcDNA3.1(+) transfected cells and pcDNA3.1(+)/CD14 transfected cells (P<0.01). The fluorescence was significantly stronger on the stable cell line Hela-CD14 than that on the transiently transfected Hela cells,and no visible fluorescence was observed in blank vector transfected cells.
  
  
  CONCLUSIONS
  The transfectant cell line Hela-CD14 with stable expression of human CD14 has been successfully established, which can be used to study human CD14 molecular and CD14-associated monocyte/macrophage cell diseases.
authors.affiliation: Department of Hematology and Oncology, Zhejiang University School of Medicine, Hangzhou, China.
authors.forename: Bo-tao
authors.initials: BT
authors.lastname: Ning
authors.forename: Hua
authors.initials: H
authors.lastname: Song
authors.forename: Shi-long
authors.initials: SL
authors.lastname: Yang
authors.forename: Wei-qun
authors.initials: WQ
authors.lastname: Xu
authors.forename: Yong-min
authors.initials: YM
authors.lastname: Tang
mesh_terms: D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014162:Transfection
chemical_list: D018950:Lipopolysaccharide Receptors