Construction of mouse CMKLR1 gene knock-down vascular smooth muscle cell lines mediated by lentivirus vector

doi:10.3969/j.issn.2095-4344.2015.20.015

Abstract：BACKGROUND:Levels of chemerin are elevated in arteriosclerosis, indicating chemerin and its receptor (CMKLR1) may participate in the pathological process of arteriosclerosis. OBJECTIVE: To establish the stable CMKLR1 gene knock-down mouse vascular smooth muscle cel lines. METHODS: Three shRNA sequences targeting the coding region of mouse CMKLR1 mRNA were synthesized and employed to construct lentivirus recombinant vectors. The best pLVX-shRNA was picked up to package recombinant lentivirus in 293T cels, which were infected in cultured mouse vascular smooth muscle cels. The CMKLR1 mRNA level of vascular smooth muscle cels was verified by real-time PCR. RESULTS AND CONCLUSION: The lentiviral vectors were successfuly constructed, which was confirmed by DNA sequencing. The titer of lentivirus reached 8.7×106 TU/mL in the packaging cels, and pLVX-shRNA3 showed the most significant interfering effects on CMKLR1 gene (P < 0.001). The pLVX-shRNA3 was chosen to establish the stable CMKLR1 gene knock-down vascular smooth muscle cel lines, in which the expression of CMKLR1 mRNA was also significantly inhibited shown on real-time PCR (P < 0.001). We finaly confirmed that the expression of CMKLR1 gene can be effectively silenced by lentivirus-mediated siRNA in mouse mouse vascular smooth muscle cels.