title: [Construction of recombinant rat δNδC/VEGF-C/Cys152Ser retrovirus vector and its expression in RAW 264.7 cells].
abstract: OBJECTIVE
  To construct a retroviral vector containing rat δNδC/VEGFand verify its expression in RAW 264.7 cells.
  
  
  METHODS
  The ddVEGF-C gene was amplified by polymerase chain reaction (PCR) from pSecTag-ddVEGF-C and cloned into pLPCX vector. After the procedure of PCR,  double enzyme digestion analysis and DNA sequencing,  the recombinant plasmid was transfected into packaging cells PT67 using Lipofectamine(TM); 2000,  and the positive clones were collected by means of puromycin selection and detected for the viral titer. The expression of ddVEGF-C mRNA and protein in the RAW264.7 cells was determined by RT-PCR and Western blotting,  respectively.
  
  
  RESULTS
  PCR and double enzyme digestion analysis demonstrated that the recombinant pLPCX-ddVEGF-C was successfully constructed by displaying two positive bands of 382 bp and 6 191 bp as expected. Viral titer was 2×10(7); CFU/mL. RT-PCR and Western blotting showed the expression of ddVEGF-C at the mRNA and protein levels in RAW 264.7 cells.
  
  
  CONCLUSIONS
  The recombinant vector pLPCX-ddVEGF-C has been successfully constructed as well as PT67 packaging cells expressing stably ddVEGF-C,  which provides a potential tool for further VEGF-C related study.
authors: Graduate School of Tianjin Medical University, Tianjin 300070, China. ghyang.cardiolab@gmail.com
authors: Guo-hong
authors: GH
authors: Yang
authors: Wen-jie
authors: WJ
authors: Ji
authors: Xin
authors: X
authors: Zhou
authors: Ying
authors: Y
authors: Bi
authors: Yu-ming
authors: YM
authors: Li
mesh_terms: D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D042582:Vascular Endothelial Growth Factor C / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D042582:Vascular Endothelial Growth Factor C