1. Gene Transduction via Lentiviral Vectors 1.Transfect 293T cells with 3 plasmids that together create non-replicating lentivirus. The lentivirus can express a marker protein (here, GFP) as well as short hairpin sequences for RNAi. 2.Concentrate virus ~100x, to obtain 10 8 -10 9 IU/ml 3.Infect target cells 4.Assess transduction by GFP fluorescence. 293T packaging cell line Non-replicating Lentivirus Target Cell Infection ‘vector’ envelope (VSV-G) gag/pol/rev

2. The Macrophage-like Cell Lines RAW264.7 and J774A.1 are Transduced by Lentivirus FSC / size RAW264.7J774A.1 FSC / size GFP fluorescence Non-infected cells Lentivirus-exposed cells 92% 84% FACS analysis 3 days post-infection:

3. Bone-Marrow Derived Macrophages are Transduced by Lentivirus Non-infected cells Lentivirus-exposed cells FACS analysis at 3 days post-infection: GFP fluorescence 42% Forward scatter (size) Side scatter (complexity)

4. Bcl-2 +/TGN B Cells are Resistant to Transduction by Lentivirus Non-stimulated Bcl-2 +/TGN B cells: Non-infected cells Lentivirus-exposed cells 3% GFP fluorescence

5. Conclusions 1.Both J774A.1 and RAW264.7 macrophage-like cell lines can be efficiently transduced by lentivirus. This will enable stable expression of short hairpin RNA (for RNAi) or expression of dominant negative proteins. 2.Bone marrow-derived macrophages can also be transduced by lentivirus. These ‘real’ macrophages can be used selectively to confirm studies in cell lines. This will also allow comparison of RNAi mediated ‘knock-downs’ with gene-targeted mouse ‘knock-outs’.