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Fig. 3. Infection of OBSCs with retroviral vectors expressing PTEN and GFP. (A-F) OBSCs derived from E14.5 embryos were infected with a retroviral construct expressing GFP (B,D,F) or they were mocked infected (A,C,E). The GFP-infected cells (B) proliferated with the same pattern as mocked infected ones (A). On average, 90% of the OBSCs were infected with the GFP-expressing vector as determined by flow cytometry (E,F). No detectable GFP was observed in the mock-infected cells (C,E). (G) OBSC cultures were infected in parallel with a vector expressing PTEN-GFP or with a vector expressing GFP. Immunoblotting with an antibody against the HA-tag confirmed that PTEN was overexpressed in infected cells. The average relative levels of PTEN were 2.2-fold greater in the PTEN-GFP infected cells than in control GFP cells (n=3). (H) OBSC cultures were infected in parallel with a vector expressing PTEN-C/S-GFP or with a vector expressing GFP. Immunoblotting with an antibody against PTEN confirmed that mutant PTEN was overexpressed in infected cells. PTEN-C/S-GFP levels were 3.1-fold those of endogenous PTEN (n=2). (I-Q). GFP infected cells were induced to differentiate by mitogen withdrawal, they were then fixed 48 hours later and the co-expression of TuJ1 and GFP (I-K) or GFAP and GFP (L-N) or O4 and GFP (O-Q) was evaluated in the cultures by double immunostaining with specific antibodies. Bar, 30 µm.
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