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. 2013 Jan 29;2(1):e68. doi: 10.1038/mtna.2012.55

Figure 2.

Figure 2

Targeted addition of the microdystrophin gene in human myoblasts. (a) Top: Schematic of IDLV donor DNA vector containing the PGK-microdystrophin-V5 expression cassette flanked by CCR5 homology arms. Bottom: Schematic of the expected result after targeted gene addition at the CCR5 locus of the microdystrophin-V5 donor DNA. PCR primers (black arrows) used for targeted integration analysis are indicated. (b) Myoblasts were transduced with the indicated viral vectors and the expression of the microdystrophin-V5 was detected by immunofluorescence using a V5-specific antibody (in red). Nuclei were counterstained with DAPI. Myoblasts were immunostained 4 weeks post-transduction to allow for the dilution of the IDLV vectors. Original magnification: ×200. (c) Quantitative representation of the levels of microdystrophin-V5 gene-targeted myoblasts 4 weeks post-transduction. The proportion of V5-positive cells was determined by counting manually a total of 300 treated cells in randomly selected fields. Results are representative of three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; IDLV, integrase-defective lentiviral vector; MOI, multiplicity of infection; NTC, non-transduced cells; PGK, phosphoglycerate kinase; ZFN, zinc finger nuclease.