All transfections were performed in 6-well plates. 293FT cells were transfected with a total of 1 μg of plasmid and 4 μg of polyethyleneimine (PEI) (30 (link)). For HIV-1-CMV-Cre particles, cells were transfected with 900 ng of provirus and 100 ng of glycoprotein expression vector (Fig. 1 ) or with 800 ng of provirus and 200 ng of glycoprotein expression vector (subsequent figures). For HIV-1-Gluc particles, cells were transfected with 800 ng of provirus and 200 ng of glycoprotein expression vector. For MLV particles, cells were transfected with 500 ng of the MLV GagPol expression vector, 400 ng of MLV-CMV-Cre, and 100 ng of glycoprotein expression vector. For VSV particles, cells were transfected with 1 μg of glycoprotein expression vector and were infected 2 days posttransfection with >107 infectious units/well of VSVΔG-GFP (Kerafast) (31 (link)). Cells were rinsed with phosphate-buffered saline (PBS) 1 h after infection, and the medium was replaced with complete medium supplemented with 2 μl of mouse hybridoma supernatant containing anti-VSV-G antibody I1 (Kerafast) to neutralize input virus. Neutralizing antibody was excluded from samples pseudotyped with VSV-G. VSV pseudoparticles were collected 24 h later.
Supernatants containing virus were frozen at –80°C for at least 4 h, thawed, and spun at 3,200 × g for 5 min, and the same volume of medium was added to target cells with 20 μg of hexadimethrine bromide per ml (H9268; Sigma). For assays with a fluorescent readout, infected cells were collected at about 2 to 3 days postinfection, fixed with 4% paraformaldehyde (PFA), washed with PBS, and analyzed on an Accuri C6 flow cytometer. For infections with a Gluc readout, transductions were allowed to proceed for 2 to 3 days, and 20 μl of supernatant from each well was transferred to a black 96-well plate for measuring Gaussia luciferase activity with 50 μl of 10 μm coelenterazine in 0.1 M Tris (pH 7.4) and 0.3 M sodium ascorbate (NanoLight Technology). Luminescence, representing infectivity, was measured from the supernatant using a PerkinElmer EnSpire 2300 multilabel reader.
Supernatants containing virus were frozen at –80°C for at least 4 h, thawed, and spun at 3,200 × g for 5 min, and the same volume of medium was added to target cells with 20 μg of hexadimethrine bromide per ml (H9268; Sigma). For assays with a fluorescent readout, infected cells were collected at about 2 to 3 days postinfection, fixed with 4% paraformaldehyde (PFA), washed with PBS, and analyzed on an Accuri C6 flow cytometer. For infections with a Gluc readout, transductions were allowed to proceed for 2 to 3 days, and 20 μl of supernatant from each well was transferred to a black 96-well plate for measuring Gaussia luciferase activity with 50 μl of 10 μm coelenterazine in 0.1 M Tris (pH 7.4) and 0.3 M sodium ascorbate (NanoLight Technology). Luminescence, representing infectivity, was measured from the supernatant using a PerkinElmer EnSpire 2300 multilabel reader.
