title: [Construction of eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 WT or its variants].
abstract: To construct eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 wild-type or its variants (W39R, R264C, W39R-R264C) and to observe its expression in MCF-7 and Bcap-37 cells.
  
  
  
  The aromatase WT cDNA sequence was obtained by RT-PCR amplification and cloned into the eukaryotic expression vector pcDNA3.1(+). pcDNA3.1(+)-CYP19-GFP plasmid was then used as the template for site-directed mutation to create variant constructs (W39R, R264C, W39R-R264C). pcDNA3.1(+)-CYP19-GFP was transfected and expressed in MCF-7 and Bcap-37 cells.
  
  
  
  The construction of pcDNA3.1(+)-CYP19-GFP plasmid was confirmed by enzyme digestion and DNA sequencing. pcDNA3.1(+)-CYP19(W39R)-GFP, pcDNA3.1(+)-CYP19(R264C)-GFP, pcDNA3.1(+)- CYP19(W39R-R264C)-GFP plasmids were confirmed by DNA sequencing. The MCF-7 and Bcap-37 cells transfected with the pcDNA3.1(+)-CYP19-GFP plasmid expressed reporter gene of GFP.
  
  
  
  The eukaryotic expression plasmids have been constructed and expressed in MCF-7 and Bcap-37 cells successfully, which lays the foundation for the research of biological activities of CYP19 variant allozymes.
authors.affiliation: Chemotherapy Center of Zhejiang Cancer Hospital, Hangzhou 310022, China. shao_xiying@yahoo.com.cn
authors.forename: Xi-Ying
authors.initials: XY
authors.lastname: Shao
authors.forename: Zhan-Hong
authors.initials: ZH
authors.lastname: Chen
authors.forename: Jiang
authors.initials: J
authors.lastname: Cao
authors.forename: Yong-Ming
authors.initials: YM
authors.lastname: Fang
authors.forename: Xiao-Jia
authors.initials: XJ
authors.lastname: Wang
mesh_terms: D001141:Aromatase / Q000235:genetics*; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001141:Aromatase