Mapping of HIV-based (LT) and MLV-based (RT) promoter traps integration site in selected 293T cells. Puromycin-resistant cell populations and clones were isolated from 293T cells transduced with LT or RT at very low vector input to ensure single-copy integration. Genomic DNA from selected bulk populations, 15 LT and 15 RT clones was subjected to vector-specific LAM-PCR to retrieve the proviral-genomic junction. Ten LT and 10 RT integrations were unambiguously mapped after BLAST analysis against the human genome database using the ENSEMBL search engine (September 2004 freeze). All characterized integrations are listed in Table 1. Three representative LT (A-C) and RT (D-F) integrations are shown here in detail. The genomic region around the integration site (length in kb indicated on top) were obtained from the ENSEMBL output and graphically simplified. Blue thick bars represent the selected chromosomal region. Query sequence from LAM-PCR product is placed in the middle of the genomic interval and is represented by a red mark (BLAST hit) above or below the chromosomal bar depending on the provirus orientation. Because LAM-PCR products from the LT and RT were obtained using the 3′ LTR and 5′ LTR as template, respectively, the orientation of the trapping cassette on the ENSEMBL output was opposite for LT and RT. For clarity, proviral integration and the direction of transcription of the Puro^R-GFP cassette are represented by a green arrow. Transcripts, protein alignments, and genomic annotations were retrieved by searching different databases as displayed on the left side of each picture. Genes displayed above the blue bar are transcribed from left to right; genes displayed below the bar are transcribed from right to left. (A) LTA4 integration on 17p13.2 landed in the first intron of the UBE2G1 gene before the first coding ATG. Promoters (circles with arrows) are identified by algorithms Eponine and FirstEF (red marks). (B) LTA3 integration on 6p21.31 landed in the third intron of the FKBP5 gene before the first coding ATG. Fusion transcript could be generated by 2 different promoters (circles with arrows) as identified by algorithms Eponine and FirstEF (red marks)¹⁹  and presence of a CpG island (purple mark). The distance from the closest promoter is 16 kb. (C) LT1 integration on 15q22.2 landed into the first intron of the BNIP gene after the first noncoding exon. Distance from transcription start site (TSS) was +5.5 kb. Reporter expression can be explained by a trap/5′ untranslated region (UTR) fusion transcript. (D) Integration RT16.2 on 20q13.32 landed into a CpG island, 350 base pair (bp) upstream of the putative transcription start site of the sintaxin 16 gene. Both First EF and Eponine algorithms identified several putative promoter regions, one of which may drive reporter expression (circle with arrow). (E) RT25 integration on 20q13.33 landed into the first intron of the GMEB2 gene after the first noncoding exon. Distance from TSS was +1 kb. First EF and Eponine algorithms identified putative promoter regions (circle with arrow). (F) RT6.1 Integration on 16p13.3 landed into the DKFZp434F054 gene. Depending on the splicing, the trap can be fused to a 5′ UTR exon. Alternatively, the same 5′ UTR portion can be spliced to generate an in-frame protein coding fusion transcript with the reporter. Distance from TSS was +0.4 kb.