Figure 2: Design and development of a template-switching lentiviral vector.
From: Lentiviral vectors can be used for full-length dystrophin gene therapy
(a) Schematic representing the pathway for reconstituting a full-length provirus from a heterozygous viral genome. Reverse-transcription proceeds until reaching a region of homology where the reverse-transcription complex can undergo template-switching and reconstitute the full-length sequence. (b) Examination of factors affecting the rate of NeoR-IRES-GFP reconstitution. Modified lentiviruses (reverse-transcriptase mutants V148I or Q151N and/or complementary DIS) were compared to unmodified vectors. GFP output was quantified by flow cytometry, where wild-type homozygous vectors were used to set the baseline for GFP expression. All samples are N = 3. *P < 0.05 by Kruskal-Wallis test with Dunn’s post-hoc analysis. Bars represent average GFP readings and standard deviation. (c) PCR analysis of NIGW reconstitution in GFP-sorted HeLa cells. Separation of products on a 1% agarose gel reveals a 3.7 kb band in the full-length NIGW positive control (2) and the wtRT.wtDIS heterozygous sample (3). 2.4 kb and 2.5 kb bands were detected in the homozygous sample (4) and the heterozygous sample. All three bands were undetectable in the non-transduced (1) and water (5) control reactions. L = NEB 2-log ladder.
