Figure 5.
Figure 5. Molecular analyses of transposition in human T cells. (A) DsRed transgene expression in transposed T-cell clones 4 months after gene transfer. (B) Schematic representation of a DsRed probe used in this study. Top row shows the circular transposon-encoded plasmid nucleofected into T cells and the location of the 735-bp DsRed probe. Bottom row shows the loss of flanking restriction enzyme recognition sequences after transposase-mediated integration into genomic DNA (wavy line). (C) Integration assay by Southern blotting. Southern blot of genomic DNA digested with SalI and XbaI and hybridized with a DsRed probe. The SalI site located outside the IR/DR is lost, resulting in the absence of a detectable 1.7-kb plasmid-specific band. Southern blot of genomic DNA from clone O56 digested with SacI and XhoI as well as KpnI and XbaI and hybridized with the same probe. The SacI and KpnI sites flanking IR/DRs are lost, resulting in the absence of detectable 2.8- and 1.7-kb plasmid-specific bands, respectively. (D) Determination of per-cell copy numbers by Southern blot. A 1-copy standard of pT2/DsRed was equal to 11 pg. Copy standards were mixed with 10 μg genomic DNA isolated from bulk activated T cells. Transgene copy numbers for each T-cell clone were calculated from the intensities of the bands spanning the DsRed gene in comparison to a copy number standard after linear regression analysis. Band intensities were quantified using a PhosphorImager. (E) Transposition assay. Transposon insertion site sequences were determined using a linker-mediated PCR technique described in “Materials and methods.” Plasmid backbone sequences of pT2/DsRed are shown. Duplicated TA dinucleotide target site is in capital letters. Transposon-specific sequences are in the center box. Amplified transposon:chromosome junction sequences were subjected to BlastN analysis against the human genome using the ENSEMBL database (chromosome locations indicated in shaded box on the left). Genome-specific primers were designed in accordance with identified chromosome positions to amplify the opposing flanking sequence and confirm insertion into a single TA dinucleotide.

Molecular analyses of transposition in human T cells. (A) DsRed transgene expression in transposed T-cell clones 4 months after gene transfer. (B) Schematic representation of a DsRed probe used in this study. Top row shows the circular transposon-encoded plasmid nucleofected into T cells and the location of the 735-bp DsRed probe. Bottom row shows the loss of flanking restriction enzyme recognition sequences after transposase-mediated integration into genomic DNA (wavy line). (C) Integration assay by Southern blotting. Southern blot of genomic DNA digested with SalI and XbaI and hybridized with a DsRed probe. The SalI site located outside the IR/DR is lost, resulting in the absence of a detectable 1.7-kb plasmid-specific band. Southern blot of genomic DNA from clone O56 digested with SacI and XhoI as well as KpnI and XbaI and hybridized with the same probe. The SacI and KpnI sites flanking IR/DRs are lost, resulting in the absence of detectable 2.8- and 1.7-kb plasmid-specific bands, respectively. (D) Determination of per-cell copy numbers by Southern blot. A 1-copy standard of pT2/DsRed was equal to 11 pg. Copy standards were mixed with 10 μg genomic DNA isolated from bulk activated T cells. Transgene copy numbers for each T-cell clone were calculated from the intensities of the bands spanning the DsRed gene in comparison to a copy number standard after linear regression analysis. Band intensities were quantified using a PhosphorImager. (E) Transposition assay. Transposon insertion site sequences were determined using a linker-mediated PCR technique described in “Materials and methods.” Plasmid backbone sequences of pT2/DsRed are shown. Duplicated TA dinucleotide target site is in capital letters. Transposon-specific sequences are in the center box. Amplified transposon:chromosome junction sequences were subjected to BlastN analysis against the human genome using the ENSEMBL database (chromosome locations indicated in shaded box on the left). Genome-specific primers were designed in accordance with identified chromosome positions to amplify the opposing flanking sequence and confirm insertion into a single TA dinucleotide.

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