Journal: Journal of Virology
Article Title: Release of Vesicular Stomatitis Virus Spike Protein G-Pseudotyped Lentivirus from the Host Cell Is Impaired upon Low-Density Lipoprotein Receptor Overexpression
Figure Lengend Snippet: Lentiviral copy number increases with decreasing amount of LDLR-encoding plasmid cotransfected with the virus-producing plasmids used in the positive control. (A) Viral RNA in the supernatant of HEK293 cells was determined by RT-qPCR. The negative controls (NegC) were psPAX2 and pMD2.G, and the positive controls (PosC) were pHR-CMV-EGFP, psPAX2, and pMD2.G. Data are the arithmetic means of 3 biological replicates ± standard errors of the means (SEM). The presence of LDLR decreases the copy number significantly [one-way analysis of variance (ANOVA), F (4, 2) = 356.18], and the copy number is inversely related to the concentration of LDLR [one-way ANOVA, F(3, 2) = 373.33]. Wedges indicate the amount of the LDLR-encoding plasmid relative to pHR-CMV-EGFP used in the transfection (LDLR, 1:2 to 1:16). ***, P
Article Snippet: To confirm the above observation and determine the underlying reason, virus was produced in HEK293 cells (2.5 × 106 /10-cm2 plate) via transient transfection with the self-inactivating vector construct pHR-CMV-EGFP (3.33 μg; Addgene 14858) , as well as the packaging construct psPAX2 (5 μg; Addgene 12260) and the envelope construct pMD2.G, encoding the VSVG surface protein (1.66 μg; Addgene 12259) (both a kind gift from Didier Trono).
Techniques: Plasmid Preparation, Positive Control, Quantitative RT-PCR, Concentration Assay, Transfection