Fig. 2.

Generation of VLPs containing transcriptional factors from HEK293T packaging cells. (a) Quantification of VLPs expression by Western blot analysis of CA. Concentrated VLPs were purified from pL-GPTP expressing cell culture (1 mL). CA was detected with a polyclonal anti-CA antibody (81S-263) and served as a loading control. Sizes for Pr-PT, and CA are indicated. O: Oct4, S: Sox2, K: Klf4, C: c-Myc, and nG: nlsGFP. M: culture supernatant from mock 293T cell. WT: wild type MLV virion. (b&c) Distribution of Gag-PT or Gag-PT-2NES in the packaging cell was analyzed by immunofluorescence. Representative images of HEK293T cells infected with lentivirus packaging pL-GPTP (panel b) or pL-GPT2NP (panel c) after selection in puromycin. Stable packaging cells were analyzed by GFP signal or immunofluorescence staining using anti-CA (Red), anti-Oct4 (Green), or anti-Sox2 (Green) antibody as described under “Materials and Methods.” Nuclei were stained with DAPI (Blue). Arrows indicate Gag chimeric protein accumulate and assemble on the inner surface of the plasma membrane. Cells were visualized with a Zeiss LSM510 META confocal microscope and bars represent 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)