Table 1. Total integration frequencies and absolute gene targeting frequencies achieved using the 2A-based or IRES-based promoter-trap system.
| Experiment | Cell line | Vector | Total integration frequencya | 2A to IRES ratio in total integration frequencyc | Absolute gene targeting frequencyb | 2A to IRES ratio in absolute gene targeting frequencyc | ||
|---|---|---|---|---|---|---|---|---|
| Mean | S.e.m. | Mean | S.e.m. | |||||
| HygR–5′ EGFP | HCT116 | IRES | 4.0 × 10−2 | 1.2 × 10−3 | 2.3 × 10−4 | 4.4 × 10−5 | ||
| 2A | 6.3 × 10−2 | 1.1 × 10−3 | 1.6 | 7.7 × 10−4 | 9.4 × 10−6 | 3.4 | ||
| DLD-1 | IRES | 1.7 × 10−2 | 1.5 × 10−4 | 1.0 × 10−3 | 8.4 × 10−5 | |||
| 2A | 3.7 × 10−2 | 4.3 × 10−4 | 2.2 | 5.3 × 10−3 | 2.1 × 10−4 | 5.1 | ||
| PIGA exon 6 | HCT116 | IRES | 4.5 × 10−3 | 2.6 × 10−4 | 1.2 × 10−4 | 3.7 × 10−5 | ||
| 2A | 8.3 × 10−3 | 3.1 × 10−4 | 1.8 | 1.2 × 10−3 | 1.4 × 10−5 | 9.6 | ||
| DLD-1 | IRES | 2.4 × 10−3 | 1.5 × 10−4 | 3.7 × 10−5 | 1.4 × 10−6 | |||
| 2A | 1.3 × 10−2 | 5.8 × 10−4 | 5.4 | 1.0 × 10−3 | 5.0 × 10−5 | 28 | ||
| AsPC-1 | IRES | 2.3 × 10−5 | 2.5 × 10−6 | 5.3 × 10−7 | 1.8 × 10−7 | |||
| 2A | 6.7 × 10−5 | 2.5 × 10−6 | 2.9 | 3.7 × 10−6 | 6.2 × 10−7 | 7.0 | ||
| PL5 | IRES | 2.8 × 10−4 | 1.4 × 10−5 | 1.9 × 10−6 | 6.6 × 10−7 | |||
| 2A | 4.4 × 10−4 | 2.1 × 10−5 | 1.6 | 1.2 × 10−5 | 7.1 × 10−7 | 6.1 | ||
| HuP-T3 | IRES | 2.3 × 10−4 | 1.1 × 10−5 | 1.2 × 10−6 | 4.4 × 10−7 | |||
| 2A | 4.0 × 10−4 | 1.9 × 10−5 | 1.8 | 5.3 × 10−6 | 8.8 × 10−8 | 4.6 | ||
| PIGA intron 5 | HCT116 | IRES | 3.5 × 10−3 | 5.1 × 10−5 | 4.5 × 10−5 | 1.7 × 10−5 | ||
| 2A | 1.3 × 10−2 | 3.6 × 10−4 | 3.6 | 6.0 × 10−4 | 6.9 × 10−5 | 13 | ||
| DLD-1 | IRES | 7.8 × 10−3 | 2.9 × 10−4 | 1.3 × 10−4 | 1.0 × 10−4 | |||
| 2A | 3.7 × 10−2 | 5.5 × 10−4 | 4.7 | 2.9 × 10−3 | 9.6 × 10−5 | 22 | ||
| + CRISPR-Cas9 | HCT116 | IRES | 1.6 × 10−2 | 2.8 × 10−4 | 4.2 × 10−3 | 3.8 × 10−4 | ||
| 2A | 7.9 × 10−2 | 6.4 × 10−4 | 5.0 | 1.8 × 10−2 | 1.4 × 10−3 | 4.3 | ||
| DLD-1 | IRES | 3.2 × 10−3 | 8.4 × 10−5 | 1.2 × 10−3 | 6.9 × 10−5 | |||
| 2A | 1.0 × 10−1 | 1.6 × 10−3 | 31 | 1.9 × 10−2 | 2.3 × 10−3 | 15 | ||
aTotal integration frequency was calculated by dividing ‘the number of G418-resistant colonies’ by ‘the number of cells inoculated for AAV infection (or plasmid transfection)’ and ‘plating efficiency’. Plating efficiencies empirically determined were 0.98 (HCT116 and its derivative), 0.87 (DLD-1 and its derivative), 0.79 (AsPC-1), 0.86 (PL5) and 0.95 (HuP-T3).
bAbsolute gene targeting frequency was calculated by multiplying total integration frequency and H/R ratio.
c‘2A to IRES ratio’ is the mean value for 2A divided by the mean value for IRES.