Figure 1

MLV vector integrations into the TNIK and LMO2 gene loci. (A) Schematic representation of MLV vector integration into a gene locus. MLV vector integrations were detected using nested PCR with a combination of 3' or 5' LTR-specific primers (3' L1L2 or 5' L1L2) and gene-specific primers (F or R). (B) MLV integration sites in the integration hotspot of the TNIK gene locus. Upper: Diagrammatic representation of the TNIK gene locus. Exons and the transcription start site are shown as Ex and +1, respectively. Lower: MLV vectors integrated into an approximately 2-kb region within the TNIK hotspot were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and TNIK-specific primers (F1F2 or R1R2), as described in (A). The numbers indicate the nucleotide distance from the TNIK-specific primers (F1F2 or R1R2). The PCR products were sequenced, and the locations of the integration sites were determined by use of the human BLAST program. The integration site within the 705-9 cell hotspot was identified in our previous study [11] (large black arrowhead). (C) MLV integration sites near exon 1 of the LMO2 gene in TPA-Mat cells. Upper: Diagrammatic representation of the LMO2 gene locus. Lower: MLV vectors integrated into an approximately. ± 3-kb region from the transcription start site of LMO2 were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and LMO2-specific primers (F3F4, F5F6, F7F8, R3R4, R5R6 or R7R8), as described in (B). The numbers indicate the nucleotide distance from the transcription start site. Pt4 and Pt5 indicate the therapeutic MLV vector integration sites in patients 4 and 5, respectively, who developed leukemia after the French X-SCID gene therapy trials. (D) MLV integration sites near exon 1 of the LMO2 gene in Jurkat-ecoR cells. (E) MLV integration sites near exon 1 of the LMO2 gene in HeLa cells. (F) MLV integration sites near exon 1 of the LMO2 gene in human CD34⁺ cells. Black and white arrowheads respectively denote the reverse and forward orientation, relative to transcription, of the integrated MLV vectors.