Transduction of SRC with lentiviral and oncoretroviral vectors
| Group . | Mouse . | Cell dose . | MOI . | TE FACS (%) . | 1° CFU-GM PCR (%) . | Primary recipients . | Secondary recipients . | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| CD45+ FACS (%) . | GFP+ FACS (%) . | 2° CFU-GM PCR (%) . | CD45+ FACS (%) . | GFP+ FACS (%) . | 3° CFU-GM PCR (%) . | ||||||
| Lentiviral vector | |||||||||||
| I | 1.1 | 100 000 | 100 | 37 | ND | 5 | 14 | ND | |||
| 1.2 | 8 | 9 | ND | ||||||||
| II | 2.1 | 100 000 | 70 | 33 | ND | 13 | 11 | ND | |||
| 2.2 | 3 | 13 | ND | ||||||||
| 2.3 | 6 | 7 | ND | ||||||||
| III | 3.1 | 400 000 | 50 | 20 | 16 | 35 | 6 | 62 (20/32) | 3 | 18 | 74 (23/31) |
| 3.2 | 37 | 10 | 80 (24/30) | 2 | 33 | 44 (7/16) | |||||
| 3.3 | 62 | 9 | 59 (19/32) | 6 | 21 | 59 (19/32) | |||||
| IV | 4.1 | 400 000 | 50 | 34 | 28 | 31 | 9 | 38 (10/26) | 4 | 27 | 71 (22/31) |
| 4.2 | 25 | 12 | 33 (7/21) | 4 | 42 | 78 (25/32) | |||||
| 4.3 | 30 | 13 | ND | Dead | |||||||
| 4.4 | 2 | 15 | 16 (4/25) | < 1 | NA | 56 (9/16) | |||||
| MGIN vector | |||||||||||
| I | 1.3 | 100 000 | 60 | < 1 | ND | 5 | < 1 | ND | |||
| 1.4 | 70 | < 1 | ND | ||||||||
| IV | 4.5 | 100 000 | 20 | < 1 | 0 | 16 | < 1 | 0 | |||
| 4.6 | 15 | < 1 | 0 | ||||||||
| 4.7 | 11 | < 1 | 0 | ||||||||
| 4.8 | 29 | < 1 | 0 | ||||||||
| Group . | Mouse . | Cell dose . | MOI . | TE FACS (%) . | 1° CFU-GM PCR (%) . | Primary recipients . | Secondary recipients . | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| CD45+ FACS (%) . | GFP+ FACS (%) . | 2° CFU-GM PCR (%) . | CD45+ FACS (%) . | GFP+ FACS (%) . | 3° CFU-GM PCR (%) . | ||||||
| Lentiviral vector | |||||||||||
| I | 1.1 | 100 000 | 100 | 37 | ND | 5 | 14 | ND | |||
| 1.2 | 8 | 9 | ND | ||||||||
| II | 2.1 | 100 000 | 70 | 33 | ND | 13 | 11 | ND | |||
| 2.2 | 3 | 13 | ND | ||||||||
| 2.3 | 6 | 7 | ND | ||||||||
| III | 3.1 | 400 000 | 50 | 20 | 16 | 35 | 6 | 62 (20/32) | 3 | 18 | 74 (23/31) |
| 3.2 | 37 | 10 | 80 (24/30) | 2 | 33 | 44 (7/16) | |||||
| 3.3 | 62 | 9 | 59 (19/32) | 6 | 21 | 59 (19/32) | |||||
| IV | 4.1 | 400 000 | 50 | 34 | 28 | 31 | 9 | 38 (10/26) | 4 | 27 | 71 (22/31) |
| 4.2 | 25 | 12 | 33 (7/21) | 4 | 42 | 78 (25/32) | |||||
| 4.3 | 30 | 13 | ND | Dead | |||||||
| 4.4 | 2 | 15 | 16 (4/25) | < 1 | NA | 56 (9/16) | |||||
| MGIN vector | |||||||||||
| I | 1.3 | 100 000 | 60 | < 1 | ND | 5 | < 1 | ND | |||
| 1.4 | 70 | < 1 | ND | ||||||||
| IV | 4.5 | 100 000 | 20 | < 1 | 0 | 16 | < 1 | 0 | |||
| 4.6 | 15 | < 1 | 0 | ||||||||
| 4.7 | 11 | < 1 | 0 | ||||||||
| 4.8 | 29 | < 1 | 0 | ||||||||
Lentiviral but not MGIN-based retroviral vectors can transduce human CD34+ cells that can repopulate primary and secondary NOD/SCID transplant recipients. The results from 4 different experiments are shown. TE indicates transduction efficiency assessed by FACS 4 days after transduction. 1° CFU-GM are colonies derived from CD34+ cells harvested directly following overnight transduction. 2° CFU-GM are colonies derived from the BM of primary recipients and 3° CFU-GM are colonies derived from the bone marrow of secondary recipients. The presence of the GFP gene was confirmed by PCR analysis of 32 individual CFU-GM colonies from 1° CFU-GM and 32 individual colonies from each recipient mouse. The large discrepancy between the percentages of vector-positive CFU-GM and GFP expressing CD45+ cells as determined by FACS may be attributed in part to the use of Becton Dickinson lysis buffer, which results in loss of GFP expression during the lysis procedure. This can be avoided by the use of ammonium chloride to lyse the red cells.
ND indicates not determined; NA, not available.