title: [Construction of human Gax gene eukaryotic expression vector and its expression in vascular smooth muscle cells].
abstract: OBJECTIVE
  To construct human Gax eukaryotic expression vector of pEGFP-N1-Gax, and observe its expression in the rabbit vascular smooth muscle cells(VSMCs).
  
  
  METHODS
  human Gax cDNA was obtained by polymerase chain reaction(PCR) from pCMV-SPORT6-Gax plasmid. After digested with Nhe I and Xho I, the PCR product was cloned into eukaryotic expression vector pEGFP-N1 of green fluorescent protein(GFP) reported gene encoding green fluorescence protein, and then the recombinant plasmid pEGFP-N1-Gax was transfected into rabbit VSMCs using Sofast after it was identified by restriction enzyme digestion analysis and gene sequencing. Human Gax expression in rabbit VSMCs was detected by examining GFP expression of transfected cells under fluorescence microscope and RT-PCR.
  
  
  RESULTS
  Agarose gel electrophoresis detection showed that human Gax DNA segment was about 915 bp, which accorded with the expectation. The restriction enzyme digestion analysis and DNA sequencing assays of recombinant vector pEGFP-N1-Gax showed the correct orientation and sequence. The expression of GFP in rabbit VSMCs transfected with pEGFP-N1-Gax was observed by fluorescence microscopy, and the expression of human Gax mRNA was confirmed by RT-PCR.
  
  
  CONCLUSIONS
  The recombinant plasmid pEGFP-N1-Gax is successfully constructed, and expressed positively in rabbit VSMCs, it provide experimental basis to study the effects of Gax gene on cardiovascular disease.
authors.affiliation: Department of Cardiothoracic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China. 123123zhenghui@163.com
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: Song
authors.initials: S
authors.lastname: Xue
authors.forename: Feng
authors.initials: F
authors.lastname: Lian
authors.forename: Yong-yi
authors.initials: YY
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary; D018398:Homeodomain Proteins; C484193:MEOX2 protein, human; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins