The pMDLg/pRRE-based viral particle production was done using pMDLg/pRRE (800 ng), pSIN.PPT.CMV.Luc.IRES.GFP (800 ng), pRSV-Rev (400 ng), and pMDG.VSV-G (200 ng). For nanoluciferase-based reporter viruses (HIV-1 N, O, P, SIVgor, and SIVcpz), 106 HEK293T cells were seeded; the following day, these cells were transfected using 200 ng VSV-G and 2,000 ng of viral plasmids using polyjet (Tebubio GmbH, Offenbach, Germany). Forty-eight hrs posttransfection, viral particles were collected. Where needed, the reverse transcriptase activity of viruses was quantified using the previously described approach (42 (link)).
Protocol detail
Viral Particle Production for Luciferase Reporters
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Corresponding Organization :
Other organizations : Heinrich Heine University Düsseldorf, Forschungszentrum Jülich
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Variable analysis
independent variables
- Amount of pMDLg/pRRE (800 ng)
- Amount of pSIN.PPT.CMV.Luc.IRES.GFP (800 ng)
- Amount of pRSV-Rev (400 ng)
- Amount of pMDG.VSV-G (200 ng)
- Amount of VSV-G (200 ng)
- Amount of viral plasmids (2,000 ng)
- Viral particle production
- Reverse transcriptase activity of viruses
- Number of HEK293T cells seeded (10^6)
- No positive or negative controls were explicitly mentioned.
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