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Gene Transduction via Lentiviral Vectors

Gene Transduction via Lentiviral Vectors. envelope (VSV-G). ‘vector’. gag/pol/rev. Transfect 293T cells with 3 pla smi ds that together create non-replicating lentivirus. The lentivirus can express a marker protein (here, GFP) as well as short hairpin sequences for RNAi.

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Gene Transduction via Lentiviral Vectors

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  1. Gene Transduction via Lentiviral Vectors envelope (VSV-G) ‘vector’ gag/pol/rev • 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 108-109 IU/ml • Infect target cells • 4. Assess transduction by GFP fluorescence. 293T packaging cell line Non-replicating Lentivirus Target Cell Infection

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

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

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

  5. Conclusions • 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. • 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’.

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