FIG. 4.

Molecular analysis of SB10 integration and expression in SB-transfected bulk T cells. (A) Genomic PCR showing that 5 of 15 bulk T cell populations transfected with SB10 or SB11 DNA plasmid were positive for transposase-encoding sequences. (B) Southern hybridization showing no detectable genomic integration of SB10 or SB11. (C) Standard curve of SB10 plasmid copy numbers by qPCR, where each point represents the mean ± SEM (n = 6 wells) of three independent assays. The standard curve for SB10 copy numbers was generated by dilution of the SB10 plasmid, ranging from 0, 0.004, 0.012, 0.037, 0.11, 0.33, 1, 3, to 9 copies. SB10 plasmid copy numbers less than 0.012 were considered not detectable and the copy number of 0.037 was not present on the linear line. Thus, the line equation established for the standard curve was based on the copy numbers ranging from 0.11, 0.33, 1, 3, to 9. (D) Copy numbers of the SB transposase in SB10⁺ or SB11⁺ bulk T cell lines and SB10⁺ clone O56. Data are presented as means ± SEM (n = 6 wells) of three independent assays. (E) RT-PCR showing no mRNA transcripts in genomic PCR-positive bulk T cells. (F) Quantitative RT-PCR showing undetectable mRNA transcript in SB10⁺ or SB11⁺ bulk T cells and an approximately 9000-fold higher expression level in O56 compared with O69. One of two representative results is shown. (G) Western blotting showing no SB10 or SB11 protein in genomic PCR-positive bulk T cells.