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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
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

the macrophage like cell lines raw264 7 and j774a 1 are transduced by lentivirus
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

bone marrow derived macrophages are transduced by lentivirus
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

bcl 2 tgn b cells are resistant to transduction by lentivirus
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

conclusions
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’.