Vector | LV2 primers (Reference) | Relative measurement GFP primers | Relative measurement WPRE primers | Relative measurement INS primers |
|---|
CMV.SIN | 1 | 1.15 ± 0.05 | 1.25 ± 0.09 | - |
s2 × 250 bp.CMV | 1 | 1.11 ± 0.02 | 1.39 ± 0.14 | - |
d2 × 250 bp.CMV | 1 | 0.69 ± 0.02 * | 0.88 ± 0.08 | - |
1.2 kb.CMV | 1 | 1.20 ± 0.05 | 1.21 ± 0.29 | 2.23 ± 0.04 * |
1.2 kb.CMV (Plasmid) | 1 | - | - | 0.98 ± 0.03 |
- Real-time PCR data showing the abundance of four sequences (see figure 1 for primer location) present in the viral vectors. 293T cells were transduced at MOI 1 (functional titer) and after 28 days cells were harvested and analysed. Abundance of sequences detected with primer pairs GFP, WPRE and INS was calculated using LV2 primers as reference. For CMV.SIN and s2 × 250 bp.CMV the ratio between LV2, GFP and WPRE was approximately 1:1:1 as expected. With respect to the d2 × 250 bp.CMV vector the ratio was roughly 1:0.7:0.9, which indicate that the GFP sequence is less abundant in cell cultures transduced with this vector (P < 0.001). This suggests that the vector undergoes some degree of rearrangement during production, reverse transcription or integration. In contrast, the 1.2 kb.CMV vector did not show any sign of rearrangements, as the ratio of LV2, GFP, WPRE and INS was approximately 1:1:1:2 when measuring on genomic DNA of transduced cells. In addition, the ratio between LV2 and INS was 1:1 when measuring on 1.2 kb.CMV plasmid DNA as expected. Significance is denoted by *.
