Home › Cell Biology › Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein
Cell Biology JoVE (Open Access) Citable · DOI

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

DOI: 10.3791/2198-v
What you'll learn
  • ✓ Design and produce Rev-dependent lentiviral vectors for HIV detection
  • ✓ Concentrate viral particles and quantify infectious titer accurately
  • ✓ Identify HIV-1-infected cells via GFP expression in living cultures
  • ✓ Apply Tat/Rev-responsive elements to visualize active viral replication
Protocol

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Difficulty
advanced
Total time
~3–5 days (transfection through titer determination and infection assay)
Model organism
HEK293 cells, primary CD4+ T cells (or permissive cell lines)
Biosafety
BSL-2

Steps

1
Transfect plasmids to produce lentiviral particles

Introduce packaging and transfer plasmids into HEK293 cells to generate Rev-dependent lentiviral vector particles. This step establishes the production system for infectious viral stocks.

▶ 01:15
2
Concentrate viral particles and determine titer

Harvest culture supernatant, concentrate lentiviral particles via ultracentrifugation or precipitation, and quantify infectious units using a standard titration assay. Accurate titer is essential for subsequent infection experiments.

▶ 04:24
3
Infect target cells with Rev-dependent lentiviral vector

Expose HIV-1-permissive or HIV-infected cells to the lentiviral vector at a defined multiplicity of infection. GFP expression will occur only in cells with active HIV Tat and Rev production.

▶ 06:15
4
Identify and analyze GFP-positive HIV-infected cells

Visualize and enumerate GFP-expressing cells using fluorescence microscopy or flow cytometry. GFP signal specifically marks cells with productive HIV-1 replication.

▶ 08:25
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