Figure 4: Packaging of full-length dystrophin into a standard lentiviral expression vector.
From: Lentiviral vectors can be used for full-length dystrophin gene therapy
(a) Schematic representing the CCL-SFFV-FLAG-Dystrophin-P2A-GFP expression cassette. The locations of primers used for provirus amplification are marked with angled red lines. (b) CCL-SFFV-Dystrophin-P2A-GFP was titred by GFP output after HEK 293T transduction. CCL-SFFV-GFP (CCL-GFP) was titred simultaneously to estimate titre-loss from dystrophin payload packaging. This comparison showed that a functional titre >1 × 106 lp/ml can be obtained from a lentivirus containing full-length dystrophin, which is 2 orders of magnitude lower than the CCL-GFP vector. Bars represent mean log titres with standard deviation from the mean. N = 3 for both samples. (c) PCR of CCL-SFFV-Dystrophin-P2A-GFP provirus from GFP-sorted HEK 293T genomic DNA. Running samples on a 1% agarose gel reveals a band of more than 10,000 base pairs in the GFP-sorted sample, which is absent from the untreated control. The expected band size for a provirus containing full-length dystrophin is 14,750 base-pairs.
