Fig. 2.
(A) 293T cells expressing two copies of GFP were infected with different lentiviral constructs, with mois as shown in B. Vector titers were determined by p24 ELISA and by TaqMan real-time PCR determination of transduced proviral genomes. On average, the vector preparations were 1.5 E4 particles per nanogram of p24. Cells were cultured for 9 days, and GFP expression levels were quantitated by FACS analysis. GFP expression levels were unaffected by infection with LV-CRE alone (Aa). Infection with LV-shGFP-OFF in the absence of LV-CRE resulted in no change in GFP levels (Ab), whereas LV-shGFP-WT and LV-shGFP-ON were equally effective in down-regulating the target (A c and d). Coinfection of LV-CRE (moi = 70) and LV-shGFP-OFF (moi = 100) resulted in efficient down-regulation of GFP expression (Ae). (B) FACS analysis composite. Coinfection of LV-CRE (moi = 70) with decreasing mois of LV-siGFP-OFF (moi = 60, 20, or 10) resulted in decreased silencing of GFP (data not shown). Similarly, decreasing of LV-CRE (moi = 70, 35, 7, and 3) while keeping LV-shGFP-OFF constant (moi = 100) diminished silencing (data not shown).
