Skip to main content
. 2021 Jan 7;13(1):75. doi: 10.3390/v13010075

Figure 3.

Figure 3

Figure 3

Lentiviral vector with ORF4 when transduced in Huh7 cell clones results in stable ORF4-expressing clones. (A) Schematic diagram of the lentiviral vector used with ORF4, GFP-tagged protein and promoter (T2A and P2A). (B) Workflow of lentiviral transduction in HEK293T cells. After 48 h of co-transfection using lipofectamine, cells were visualized for GFP expression. Supernatants were harvested and used to infect Huh7 S10-3 cells. Clonal cell lines were prepared using limiting dilution method. (C) Fluorescence microscope image illustrating GFP-positive cells which suggests the successful transduction of the lentiviral vector in both the cell lines (Empty = ORF4− and ORF4 = ORF4+). (D) Agarose gel electrophoresis after RT-PCR utilizing ORF4 specific PCR primers indicates the presence of ORF4 RNA and absence of ORF4 RNA in the transduced Huh7 ORF4+ and ORF4− cell clones, respectively. Each band represents ORF4 RNA of 500 bp. CL stands for clones that were formed after the limiting dilution. ORF4+ CL (3, 4, 5) represents the ORF4+ cell lines. ORF4−CL 4 represents the ORF4− cell line. (E) Western blots of Huh7 ORF4+ cell clones (3, 4, 5) and Huh7 ORF4− cell clone 1 lysates transiently expressing the HEV ORF4 protein of 20 kDa. The blot was probed with goat anti-rabbit IgG, horse radish peroxidase to detect the expression of the HEV ORF4 protein.