Table 1.
Vector copy number estimation in PLB cells
| Samplea | Provirus | Reaction type | Probe | Copies/μl | VCNb |
|---|---|---|---|---|---|
| SC91 | pAlpha.SIN.EFS.gp91s.oPRE | Singleplex | CYBB (CO) | 36.4 | 1.10 |
| PURO | 0.03 | 0.00 | |||
| RPP30 | 66.2 | NA | |||
| Multiplex | CYBB (CO) | 36.7 | 1.10 | ||
| RPP30 | 66.5 | NA | |||
| Multiplex | PURO | 0.04 | 0.00 | ||
| RPP30 | 61.1 | NA | |||
| E91P | pAlpha.SIN.EFS.gp91s.IRES.PURO.oPRE | Singleplex | CYBB (CO) | 23.2 | 1.12 |
| PURO | 24.3 | 1.17 | |||
| RPP30 | 41.6 | NA | |||
| Multiplex | CYBB (CO) | 23.9 | 1.24 | ||
| RPP30 | 38.7 | NA | |||
| Multiplex | PURO | 24.4 | 1.20 | ||
| RPP30 | 40.6 | NA | |||
| S91P | pAlpha.SIN.SFFV.gp91s.IRES.PURO.oPRE | Singleplex | CYBB (CO) | 16.7 | 1.12 |
| PURO | 18.4 | 1.23 | |||
| RPP30 | 29.8 | NA | |||
| Multiplex | CYBB (CO) | 16.3 | 1.25 | ||
| RPP30 | 26.0 | NA | |||
| Multiplex | PURO | 17.4 | 1.25 | ||
| RPP30 | 27.9 | NA |
CYBB (CO), codon-optimized CYBB (cytochrome b558 subunit β); NA, not applicable; PURO, puromycin; RPP30, ribonuclease P protein subunit p30; SFFV, spleen focus-forming virus; VCN, vector copy number.
a
SC91, the PLB clone having the indicated provirus as a single copy, which contains CYBB but no PURO sequence; E91P, the bulk PLB cell population transduced with the indicated viral vector (E stands for the EFS promoter whereas P indicates the presence of PURO); S91P, the bulk PLB cell population transduced with the indicated vector (S stands for SFFV whereas P stands for PURO).
b
VCN was calculated by the formula: CYBB or PURO copies/RPP30 copies × 2.