Retroviral transduction efficiency and vector copy numbers in mass cultures and cell clones derived thereof
Cell groups and clones . | MOI (× no. of preloading cycles) . | Gene transfer, % . | Vector copy no., real-time PCR . | Vectors/transduced cell, calculated . | MF, arbitrary units . | Average MF ± SD (variation factor)‡ . | Vector copies, Southern/qPCR . | Mean copy no./transduced cell ± SD (range)‡ . | MF/vector copy, arbitrary units (mean) . |
|---|---|---|---|---|---|---|---|---|---|
| K562 A | 0.5 | 28.6 | 0.24 | 0.9 | 33.3 ± 8.9 (1.9×) | 1.0 ± 0.0 (1) | (33.3) | ||
| 1 | 40.9 | 1 | 40.9 | ||||||
| 5 | 43.6 | 1* | 43.6 | ||||||
| 6 | 23.7 | 1* | 23.7 | ||||||
| 8 | 32.5 | 1 | 32.5 | ||||||
| 9 | 25.6 | 1 | 25.6 | ||||||
| K562 B | 2.5 | 60.9 | 1.78 | 2.9 | 43.5 ± 28.5 (6.5×) | 2.0 ± 0.8 (1-3) | (20.2) | ||
| 1 | 22.9 | 2* | 11.5 | ||||||
| 7 | 44.7 | 2 | 22.4 | ||||||
| 8 | 86.8 | 3 | 28.9 | ||||||
| 9 | 13.4 | 1 | 13.4 | ||||||
| 10 | 49.8 | 2 | 24.9 | ||||||
| K562 C | 5 | 78.0 | 2.89 | 3.7 | 162.0 ± 121.9 (14.2×) | 5.4 ± 3.7 (1-9) | (28.1) | ||
| 4 | 21.7 | 1 | 21.7 | ||||||
| 5 | 177.1 | 7* | 25.3 | ||||||
| 6 | 57.1 | 2* | 28.6 | ||||||
| 10 | 245.2 | 8 | 30.6 | ||||||
| 15 | 308.9 | 9 | 34.3 | ||||||
| K562 D | 20 | 70.3 | 6.27 | 8.9 | 162.9 ± 174.9 (29×) | 5.8 ± 6.0 (1-16) | (24.9) | ||
| 1 | 38.5 | 2 | 19.3 | ||||||
| 4 | 449.1 | 16 | 28.12 | ||||||
| 5 | 15.5 | 1 | 15.5 | ||||||
| 6 | 195.0 | 6 | 32.5 | ||||||
| 7 | 116.5 | 4* | 29.1 | ||||||
| K562 E | (2 ×) 5 | 93.5 | 8.90 | 9.5 | 251.2 ± 247.9 (19×) | 7.6 ± 5.5 (1-15) | (31.2) | ||
| 2 | 301.4 | 11 | 27.4 | ||||||
| 4 | 85.0 | 6 | 14.2 | ||||||
| 9 | 179.4 | 5* | 35.9 | ||||||
| 10 | 655.8 | 15* | 43.7 | ||||||
| 11 | 34.6 | 1 | 34.6 | ||||||
| K562 F | (2 ×) 20 | 89.6 | 9.07 | 10.1 | 752.9 ± 787.1 (17×) | > 16.0 ± 10.0 (4->31) | (< 37.1) | ||
| 1 | 860.3 | 19 | 35.8 | ||||||
| 5 | 367.5 | 14 | 26.2 | ||||||
| 8 | 339.1 | 13* | 26.1 | ||||||
| 10 | 2075.3 | > 31† | < 66.9 | ||||||
| 12 | 122.1 | 4 | 30.5 | ||||||
| CD34 A | 0.2 | 4.7 | 0.04 | 0.9 | ND | NA | ND | NA | NA |
| CD34 B | 1 | 31.6 | 0.39 | 1.2 | ND | NA | 1.6 ± 0.5 (1-2) | NA | |
| 6 | 2 | ||||||||
| 8 | 1 | ||||||||
| 10 | 1 | ||||||||
| 17 | 2 | ||||||||
| 18 | 2 | ||||||||
| CD34 C | 2 | 42.5 | 0.92 | 2.2 | ND | NA | 2.0 ± 1.2 (1-4) | NA | |
| 1 | 2 | ||||||||
| 6 | 2 | ||||||||
| 12 | 1 | ||||||||
| 13 | 1 | ||||||||
| 16 | 4 | ||||||||
| CD34 D | 4 | 58.7 | 1.01 | 1.7 | ND | NA | 2.0 ± 1.0 (1-3) | NA | |
| 3 | 2 | ||||||||
| 4 | 1 | ||||||||
| 6 | 1 | ||||||||
| 9 | 3 | ||||||||
| 12 | 3 | ||||||||
| CD34 E | (2 ×) 2 | 52.8 | 1.79 | 3.4 | ND | NA | 3.0 ± 1.2 (2-5) | NA | |
| 4 | 3 | ||||||||
| 5 | 3 | ||||||||
| 11 | 2 | ||||||||
| 16 | 5 | ||||||||
| 18 | 2 | ||||||||
| CD34 F | (2 ×) 4 | 67.2 | 2.79 | 4.2 | ND | NA | 6.0 ± 8.5 (1-21) | NA | |
| 1 | 4 | ||||||||
| 3 | 2 | ||||||||
| 6 | 2 | ||||||||
| 11 | 1 | ||||||||
| 12 | 21 |
Cell groups and clones . | MOI (× no. of preloading cycles) . | Gene transfer, % . | Vector copy no., real-time PCR . | Vectors/transduced cell, calculated . | MF, arbitrary units . | Average MF ± SD (variation factor)‡ . | Vector copies, Southern/qPCR . | Mean copy no./transduced cell ± SD (range)‡ . | MF/vector copy, arbitrary units (mean) . |
|---|---|---|---|---|---|---|---|---|---|
| K562 A | 0.5 | 28.6 | 0.24 | 0.9 | 33.3 ± 8.9 (1.9×) | 1.0 ± 0.0 (1) | (33.3) | ||
| 1 | 40.9 | 1 | 40.9 | ||||||
| 5 | 43.6 | 1* | 43.6 | ||||||
| 6 | 23.7 | 1* | 23.7 | ||||||
| 8 | 32.5 | 1 | 32.5 | ||||||
| 9 | 25.6 | 1 | 25.6 | ||||||
| K562 B | 2.5 | 60.9 | 1.78 | 2.9 | 43.5 ± 28.5 (6.5×) | 2.0 ± 0.8 (1-3) | (20.2) | ||
| 1 | 22.9 | 2* | 11.5 | ||||||
| 7 | 44.7 | 2 | 22.4 | ||||||
| 8 | 86.8 | 3 | 28.9 | ||||||
| 9 | 13.4 | 1 | 13.4 | ||||||
| 10 | 49.8 | 2 | 24.9 | ||||||
| K562 C | 5 | 78.0 | 2.89 | 3.7 | 162.0 ± 121.9 (14.2×) | 5.4 ± 3.7 (1-9) | (28.1) | ||
| 4 | 21.7 | 1 | 21.7 | ||||||
| 5 | 177.1 | 7* | 25.3 | ||||||
| 6 | 57.1 | 2* | 28.6 | ||||||
| 10 | 245.2 | 8 | 30.6 | ||||||
| 15 | 308.9 | 9 | 34.3 | ||||||
| K562 D | 20 | 70.3 | 6.27 | 8.9 | 162.9 ± 174.9 (29×) | 5.8 ± 6.0 (1-16) | (24.9) | ||
| 1 | 38.5 | 2 | 19.3 | ||||||
| 4 | 449.1 | 16 | 28.12 | ||||||
| 5 | 15.5 | 1 | 15.5 | ||||||
| 6 | 195.0 | 6 | 32.5 | ||||||
| 7 | 116.5 | 4* | 29.1 | ||||||
| K562 E | (2 ×) 5 | 93.5 | 8.90 | 9.5 | 251.2 ± 247.9 (19×) | 7.6 ± 5.5 (1-15) | (31.2) | ||
| 2 | 301.4 | 11 | 27.4 | ||||||
| 4 | 85.0 | 6 | 14.2 | ||||||
| 9 | 179.4 | 5* | 35.9 | ||||||
| 10 | 655.8 | 15* | 43.7 | ||||||
| 11 | 34.6 | 1 | 34.6 | ||||||
| K562 F | (2 ×) 20 | 89.6 | 9.07 | 10.1 | 752.9 ± 787.1 (17×) | > 16.0 ± 10.0 (4->31) | (< 37.1) | ||
| 1 | 860.3 | 19 | 35.8 | ||||||
| 5 | 367.5 | 14 | 26.2 | ||||||
| 8 | 339.1 | 13* | 26.1 | ||||||
| 10 | 2075.3 | > 31† | < 66.9 | ||||||
| 12 | 122.1 | 4 | 30.5 | ||||||
| CD34 A | 0.2 | 4.7 | 0.04 | 0.9 | ND | NA | ND | NA | NA |
| CD34 B | 1 | 31.6 | 0.39 | 1.2 | ND | NA | 1.6 ± 0.5 (1-2) | NA | |
| 6 | 2 | ||||||||
| 8 | 1 | ||||||||
| 10 | 1 | ||||||||
| 17 | 2 | ||||||||
| 18 | 2 | ||||||||
| CD34 C | 2 | 42.5 | 0.92 | 2.2 | ND | NA | 2.0 ± 1.2 (1-4) | NA | |
| 1 | 2 | ||||||||
| 6 | 2 | ||||||||
| 12 | 1 | ||||||||
| 13 | 1 | ||||||||
| 16 | 4 | ||||||||
| CD34 D | 4 | 58.7 | 1.01 | 1.7 | ND | NA | 2.0 ± 1.0 (1-3) | NA | |
| 3 | 2 | ||||||||
| 4 | 1 | ||||||||
| 6 | 1 | ||||||||
| 9 | 3 | ||||||||
| 12 | 3 | ||||||||
| CD34 E | (2 ×) 2 | 52.8 | 1.79 | 3.4 | ND | NA | 3.0 ± 1.2 (2-5) | NA | |
| 4 | 3 | ||||||||
| 5 | 3 | ||||||||
| 11 | 2 | ||||||||
| 16 | 5 | ||||||||
| 18 | 2 | ||||||||
| CD34 F | (2 ×) 4 | 67.2 | 2.79 | 4.2 | ND | NA | 6.0 ± 8.5 (1-21) | NA | |
| 1 | 4 | ||||||||
| 3 | 2 | ||||||||
| 6 | 2 | ||||||||
| 11 | 1 | ||||||||
| 12 | 21 |
CD34+ and K562 cells were transduced with SFα11eGFP17 at different MOIs (column 2). In columns 3 to 5, data for mass culture infections are given. Results represent 1 of 2 nearly identical duplicates. Five clones were derived from each mass culture (column 1) and subsequently analyzed for transgene expression (columns 6 and 7) and insertion (columns 8 and 9). See “Study design” regarding calculations of the given MOIs. The variation factors in the seventh column represent the variation factor between the lowest and the highest value in a given group. MF indicates mean fluorescence; qPCR, quantitative PCR; ND, not determined; and NA, not applicable.
For the 9 K562 clones, quantitative data were obtained by Southern blot analysis and real-time PCR, and a standard curve was generated allowing us to determine transgene copy numbers in the mass cultures
For clone 10 in K562 group F, the copy number was above the resolution limit of the Southern blot
Values in this column were calculated for the 5 clones shown in each cell group