Figure 2.

Generation of cTag8-Expressing 293T Cells for Modified Vector Production

(A) Both 293T (non-transduced) and 293T cells expressing cTag8 (cTag8 293T) by γ-retroviral transduction with cTag8 co-expressed with EGFP were stained with streptavidin-APC for cTag8 expression analysis by flow cytometry. (B) Surface expression analysis of cTag8 by immunofluorescence staining of cTag8 293T cells with streptavidin-APC. Engineered cells were assessed for LV packaging capacity by the production of LVs from both 293T cells (non-modified, NM LVs) and cTag8 293T cells (cTag8 LVs) in plain DMEM, pseudotyped with either RDpro, MLV-ampho, or VSV-G glycoproteins. (C) Viral supernatants were frozen, viral titers (infectious units (IU)/mL) of stocks were determined by infectivity assay, and mean values are presented ± SD of triplicate determinations. (D) Sucrose-cushion-ultracentrifuge-purified VSV-G pseudotyped NM LVs and cTag8 LVs were negatively stained and analyzed by transmission electron microscopy (TEM). Scale bars, 200 nm.