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. 2014 Feb 20;21(4):402–412. doi: 10.1038/gt.2014.11

Figure 2.

Figure 2

Identification of oligoclonal CLECs with transgene integration at 8p22 and FVIII secretion. (a) Oligoclonal cells (four flow-sorted cells per well) from a bulk population of G418-selected CLECs electroporated with pattBhybrid FVIII and phiC31 integrase were investigated by direct in situ PCR for transgene integration at the 8p22 hot spot. In situ lysed cells were screened using Phusion human specimen direct PCR kit (Thermo Scientific) and primers specific for vector and genomic sequences at the 8p22 integration site to detect the presence of left and right integration junctions. Intron-spanning vector-specific primers were used to amplify C1 domain of the integrated FVIII cDNA (FVIII vector). Control genomic PCR amplified a 900-bp region in 19q13.42 (AAVS1 locus). ‘–ve' denotes minus template PCR amplification while ‘+ve' denotes amplification from genomic DNA isolated from a previously identified clonal CLEC with integration at 8p22. Amplified products were electrophoresed on 1% agarose gels and imaged using BioRadGel Doc 2000 transilluminator and QuantityOne software. Red box highlights samples, which were positive for left and right integration junctions, FVIII vector and control PCR. (b) Genome-modified oligoclonal CLECs identified by junction PCR to be positive for transgene integration at chromosome 8p22 were screened by FISH. Fluorescence images of 4,6-diamino-2-phenylindole (DAPI)-stained cells hybridized with fluorescein isothiocyanate (FITC)-labelled probes specific to integrated vector (green signal) and Texas Red-labelled centromeric probes specific to chromosome 8 (red signal) are shown (original magnification × 600). Integration of transgene at chromosome 8 is indicated by the close proximity of the vector-specific green signal and chromosome 8 centromere red signal. (c) CLECs that were electroporated without any plasmid DNA (EP only) and oligoclonal CLECs that were positive for transgene integration at 8p22 (10, 15, 16, 28, 39, 40, 50, 59, 62, 68 and 69) and three oligoclonal populations that were negative for 8p22 transgene integration (18, 30 and 47) were evaluated for FVIII secretion (day 40 post-electroporation). Overnight conditioned media was assayed for FVIII activity using a Coamatic FVIII kit (Chromogenix). Data are mean±s.e.m.; n=3 per group.