title: [Construction and assessment of short-hairpin RNA eukaryotic expression vector targeting TGF-beta1 labeled by GFP].
abstract: OBJECTIVE
  To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-beta1 for further research on the effects of TGF-beta1 on vasculogenesis and angiogenesis.
  
  
  METHODS
  Three pairs of siRNA target sequences coding from the mRNA of TGF-beta1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, the three resulting TGF-beta1 shRNA expression vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-beta1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression.
  
  
  RESULTS
  RT-PCR and Western blot showed that one of the TGF-beta1 shRNA expression vectors pDS_Tc downregulated TGF-pl mRNA and protein expression markedly in NIH/3T3 cells.
  
  
  CONCLUSIONS
  ShRNA eukaryotic expression vectors targeting TGF-beta1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-beta1 regulates vasculogenesis and angiogenesis.
authors.affiliation: Department of Cardiology, Shenyang General Hospital, Cardiovascular Research Institute of PLA, Shenyang 110016, China.
authors.forename: Ya-ling
authors.initials: YL
authors.lastname: Han
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Kang
authors.forename: Yan-mei
authors.initials: YM
authors.lastname: Qi
authors.forename: Liang
authors.initials: L
authors.lastname: Guo
authors.forename: Cheng-hui
authors.initials: CH
authors.lastname: Yan
mesh_terms: D000818:Animals; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D018919:Neovascularization, Physiologic; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D049452:Green Fluorescent Proteins