title: [The construction and expression of PcDNA3.1(+)/GFP-TIMP-2 in human ameloblastoma cell].
abstract: OBJECTIVE
  To construct the eukaryotic expression vector of TIMP-2 gene and to explore its expression in human ameloblastoma cell in vitro.
  
  
  METHODS
  The aimed gene fragment was obtained by RT-PCR. And then, molecmicrolar cloning technology and enzyme digestion were used to connect the gene with the plasmid PcDNA3.1(+), which can be expressed in eukaryotic cells and a report gene: green fluorescent protein gene (GFP) was already existed in the plasmid. We named the eukaryotic expression vector, which contended our aimed gene TIMP-2 as well as report gene GFP, PcDNA3.1(+)/GFP-TIMP-2. The vector was identified by PCR analysis, EcoR I and Xho I restriction analysis and Sequence analysis. After the PcDNA3.1(+)/GFP-TIMP-2 was transfected into cultured human ameloblastoma cell, RT-PCR and Flow Cytometry (FCM) and Microscope wre respectively performed to evaluate the effect of transfection and expression.
  
  
  RESULTS
  The constructed vector PcDNA3.1(+)/GFP-TIMP-2 was proved correct by enzyme digestion and sequencing analysis. After PcDNA3.1(+)/GFP-TIMP-2 was trasnfected into cultured human ameloblastoma cell, the rate of transfection is 47.6% (Analysis report of FCM), the green fluorescence was found in plasm (observed with fluo-microwave), the expression of TIMP-2 mRNA was elevated 2.4 times compared with the control group.
  
  
  CONCLUSIONS
  PcDNA3.1(+)/GFP-TIMP-2 was successfully constructed and it could be transfected into cultured human ameloblastoma cell. It may be benefit to further study of the relationship between the TIMP-2 gene and the behaviour of ameloblastoma.
authors.affiliation: Department of Oral and Maxillofacial Surgery, The Second Affiliated Hospital, Sun Yet-sen University, Guangzhou 510120, China. huanghongzhang@tom.com
authors.forename: Hong-zhang
authors.initials: HZ
authors.lastname: Huang
authors.forename: Lei-tao
authors.initials: LT
authors.lastname: Zhang
authors.forename: Dong-lin
authors.initials: DL
authors.lastname: Zeng
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000564:Ameloblastoma / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007573:Jaw Neoplasms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D049452:Green Fluorescent Proteins