TABLE 2.
Transduction of various cell lines by HIV-1 vectors encoding GFP under the control of different promoters
| Vector (p24 level) | Mean ± SD of GFCa/ml or GFMIb/cell in transduced cell line:
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| D17
|
293
|
HeLa
|
THP-1
|
3T3
|
||||||
| GFC/ml | GMFI/cell (relative FI)d | GFC/ml | GMFI/cell (relative FI) | GFC/ml | GMFI/cell (relative FI) | GFC/ml | GMFI/cell (relative FI) | GFC/ml | GMFI/cell (relative FI) | |
| Mock | 0 | 2 ± 0 | 0 | 1c | 0 | 1 ± 1 | 0 | 2c | 0 | 2 ± 0 |
| pN-GIT72 (5.5 ng/ml) | (1.1 ± 0.2) × 105 | 1,032 ± 31 (100) | 9.6 ± 0.4 × 104 | 834 ± 21 (100) | 5.4 ± 0.1 × 105 | 607 ± 23 (100) | 6.9 ± 2.4 × 103 | 71c (100) | 1.4 ± 0.1 × 105 | 13 ± 0 (100) |
| pN-sCMVGFP (1.3 ng/ml) | (2.1 ± 0.2) × 104 | 160 ± 23 (15.5) | 1.4 ± 0.4 × 105 | 30 ± 0 (3.6) | 1.6 ± 0.1 × 105 | 171 ± 8 (28.2) | 2.4 ± 0.1 × 103 | 14 ± 0 (19.7) | 9.5 ± 0.8 × 104 | 27 ± 0 (207.7) |
| pN-SVGFP (4.1 ng/ml) | (1.5 ± 0.4) × 104 | 105 ± 8 (10.2) | 7.7 ± 1.3 × 104 | 8 ± 0 (1.0) | 1.8 ± 0.1 × 105 | 31 ± 0 (5.1) | 1.6 ± 0.2 × 103 | 18 ± 1 (25.4) | 1.1 ± 0.1 × 105 | 22 ± 0 (169.2) |
Indicated cell lines were transduced with 0.5 ml of each virus stock as described previously (64). The cells in each well were trypsinized, quenched with serum-containing medium, and washed with phosphate-buffered saline (PBS). After treatment with 4% paraformaldehyde for 30 min at room temperature, the cells were washed with PBS and resuspended in 500 μl of PBS before analysis by flow cytometry. The GFC concentration was estimated by using the formula [(no. of GFP-positive cells/volume analyzed by flow cytometry) × total cell suspension volume] × dilution factor of virus stock. The titers were corrected for background (derived from mock-transduced cells). The titer is probably overestimated by a factor of 2 to 4 due to several cell divisions between the time of transduction and the time of harvest (approximately 60 h). Data were derived from duplicate experiments.
Determined from the GFP-positive fraction (gated based on the signal obtained from mock-transduced cells.) The GMFI of mock-transduced cells was derived from control cells gated only to eliminate dead cells and debris. Values have been rounded off to the nearest whole number.
Single determination.
Relative fluorescence intensity (FI) was calculated using the formula (GMFI of each indicated vector/GMFI of pN-GIT72) × 100.