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Published on: December 3, 2013
[Construction of recombinant rat δNδC/VEGF-C/Cys152Ser retrovirus vector and its expression in RAW 264.7 cells]
Guo-hong Yang1, Wen-jie Ji, Xin Zhou
1Graduate School of Tianjin Medical University, Tianjin 300070, China. ghyang.cardiolab@gmail.com
Aim:
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.
Conclusion:
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.
