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. 2015 May 12;23(6):1110–1122. doi: 10.1038/mt.2015.48

Figure 6.

Figure 6

Integration and clonal selection analyses. (a) Schematic showing location of primers and restriction sites in the integrated virus. Clonal dominance was explored using a rapid modified LM-PCR assay (Supplementary Methods). The relative number of individual clones present was assessed by ligating Mse1-specific adaptors (M-A) to digested DNA (Supplementary Methods), then using PCR with nested primer pairs that bind to the adaptors (filled arrowheads, (Supplementary Table S2)) and the 3' end of the virus long terminal repeat (LTR, arrows). An internal control product (IC, 563 bp) is made from all TdT after a single digest with MseI (M), and provides an indication of the relative number of TdT in the sample. The internal control cannot be made after a double digest with Mse 1 and Pst1 (P), confirming specificity of the PCR reaction. To eliminate random variability in PCR, both the digests and the reactions were done in duplicate during validation and in triplicate on patient samples. To be considered prominent, a band had to occur in all replicates. (b,c) LM-PCR to distinguish non-Td NIH 3T3 cells from clones with one 350bp integration, (marked by square, ~350 bp, clone 1.5) or two (~125 and 175 bp, clone 2.5). Mix of cells shows all three products. NTC, no template control. MW = 100 bp molecular weight ladder (dots). (d) Control used primers that amplify a muGAPDH product (filled triangle, 248 bp). (e) Representative patient TK01: non-Td and Td donor cells versus patient peripheral blood mononuclear cells collected at baseline and 2 weeks after donor lymphocyte infusion. A smear or multiple bands are observed if a population of cells has many different integrations. Samples were rerun on more highly resolving gels if needed to confirm or refute identity between replicates. Dots = 50 bp molecular weight ladder. (f) Using primers to detect huGAPDH, both the functional gene (filled triangle, 234 bp) and two huGAPDH pseudogenes (open triangles, ~520 and ~130 bp) are detected as expected based on primer sequence.