Tre-mediated provirus excision and determination of Tre efficacy.

(A) Schematic depiction of Tre-mediated recombination. The PCR primers P1 and P2 amplify sequences from the provirus into the LTRs. Site-specific Tre-mediated recombination leaves a single LTR in the genome (“genomic scar”) and excises a circular recombination product containing the P1 and P2 primer binding regions. (B) Genomic DNA isolated from HeLa-smurf cells transduced with LV-Tre or LV-Ctr was analyzed by PCR using the P1 and P2 primers to detect the circular recombination product (1 kb). Negative PCR control, mock; lane M: DNA marker; p.t., post transduction. (C) To detect the genomic scar, genomic DNA prepared from Tre-treated (Tre +) or Ctr-treated cells (Tre −) 3 days after transduction were used as templates for PCR using HiLo PCR. Negative PCR control, mock; lane M: DNA marker. (D) The LTR region in the circular recombination product was subjected to DNA sequencing, revealing the presence of a single LTR flanked by nef-derived and gag leader sequences. Tre-treatment resulted in precise loxLTR recombination (boxed). (E) Genomic DNA containing either a single full length proviral genome or the residual LTR (“genomic scar”) is subjected to nrLAM PCR using LTR-specific primers (LTRIII). Subsequently, LAM PCR products consisting of LTR and genomic host DNA (LTR/int; 146 bp) or LTR and proviral coding sequence (LTR/blb; 150 bp) were quantified by qPCR. (F) Semi-quantitative PCR analysis of LAM PCR products from Tre-treated cells (Tre +), Ctr-treated control cells (Tre −) or negative PCR control (Ø). Lane M: DNA marker. (G) Quantitative PCR determination of LTR/int and LTR/blb LAM PCR products from Tre-treated cells (Tre +) or Ctr-treated (Tre −) control cells. Given are the means of three independent PCR reactions.