title: [Construction of recombinant lentiviral vector of Tie2-RNAi and its influence on malignant melanoma cells in vitro].
abstract: OBJECTIVE
  To construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells.
  
  
  METHODS
  Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency.
  
  
  RESULTS
  The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P<0.01). There was no significant difference in the expression level (P>0.05) between the two lentiviral vectors of Tie2-RNAi.
  
  
  CONCLUSIONS
  Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.
authors.affiliation: Department of Plastic Surgery, First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China.
authors.forename: Xiu-ying
authors.initials: XY
authors.lastname: Shan
authors.forename: Zhao-liang
authors.initials: ZL
authors.lastname: Liu
authors.forename: Biao
authors.initials: B
authors.lastname: Wang
authors.forename: Guo-xiang
authors.initials: GX
authors.lastname: Guo
authors.forename: Mei-shui
authors.initials: MS
authors.lastname: Wang
authors.forename: Fu-lian
authors.initials: FL
authors.lastname: Zhuang
authors.forename: Chuan-shu
authors.initials: CS
authors.lastname: Cai
authors.forename: Ming-feng
authors.initials: MF
authors.lastname: Zhang
authors.forename: Yan-ding
authors.initials: YD
authors.lastname: Zhang
mesh_terms: D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008545:Melanoma / Q000235:genetics*; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D042787:Receptor, TIE-2 / Q000235:genetics*; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; D042787:Receptor, TIE-2