Construction Of Receptor Of OxLDL Gene Silencing Cell Lines By Lentiviral-Mediated RNA Interference Technology

Object:To study effect of Lox-1 on atherosclerosis at the early stage, by establishing a silent cell model for Lox-1 to investigate the interaction between Lox-1 and its ligand and the mechanism involved in formation.Methods:Mouse Lox-1 gene fragment was amplified from J774 a.1 cell’s tototal by PCR and subcloned into psi CHECK2 plasmid. Design LOX-1 silent sh RNA and then screened the useful silent sh RNA by luciferase’ activity. The recombinant plasmid with the package plasmid transfected into 293 T cells by lipidosome, and cultured the cell for 48 h to collect the recombinant virus. Detect the titer of the recombinant virus,and then use the virus to infect J774 a.1. At last,detect LOX-1-m RNA and protein of the silent J774 a.1Results:The cloned ORF of Lox-1 gene fragment was confirmed by DNA sequencing and was subcloned into the psi CHECK2 plasmid. Design LOX-1 silent sh RNA and then screened out the useful silent sh RNA by luciferase’ activity.After packaging the lentiviral by co-transfection and concentratation of lentiviral,We harvested the virus suspension titered 4×107/m L.At last,Lentiviral particles were successfully infected J774 a.1 cells.Conclusion:We successfully construct a stable and efficient LOX-1 gene silencing cell line by lentiviral-mediated RNA interference.