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Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

Figure 3

Effect of multiple components on expression of individual anti-HIV transgenes.

(A) Expression of the membrane-bound C46 fusion inhibitor was detected by antibody staining in transduced MAGI-CCR-5 cells. Cells transduced with either a control vector RSC-SMPGW that does not express C46, or the C46 vector RSC-SC46-IMPGW, or the U6-driven site I shRNA and C46 vector RSC-UsI-SC46-IMPGW were sorted to over 98% EGFP-positive cells and stained using a primary C46 monoclonal and a secondary phycoerythrin-conjugated antibody. Expression of the C46 transgene is greatly reduced in cells transduced with the vector that also expresses the U6-driven site I tat/rev shRNA. (B) Expression of the U6-driven site I tat/rev shRNA was compared by Northern blot in MAGI-CCR-5 cells using a tat/rev site I-specific probe (top panel). NTC is non-transduced control, EGFP is cells transduced with a lentiviral vector that only expressed EGFP, UsI is cells transduced with a vector that expresses EGFP and the U6-driven site I shRNA, C46 is the vector RSC-SC46-IMPGW, UsI+C46 is the vector RSC-UsI-SC46-IMPGW. The arrow indicates approximately 21 nt. Expression of the U6-driven site I tat/rev shRNA was not affected by the addition of a C46 transgene. The bottom panel is a loading control with a U6 snRNA probe hybridized to the same blot.

Figure 3