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Hot Spots for Gene-Trap Insertion?

Hot Spots for Gene-Trap Insertion?. Agenda. Gene trap mutagenesis Gene trap sequence tag alignment Uses of gene trap tag alignments Summary of alignments in GUS Distribution of alignments along genome Hot spots of alignment/insertion Future plans. Infection or electrporation.

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Hot Spots for Gene-Trap Insertion?

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  1. Hot Spots for Gene-Trap Insertion?

  2. Agenda • Gene trap mutagenesis • Gene trap sequence tag alignment • Uses of gene trap tag alignments • Summary of alignments in GUS • Distribution of alignments along genome • Hot spots of alignment/insertion • Future plans

  3. Infection or electrporation Selection with drug Injection into embryo

  4. Gene Trap Mutagenesis Retroviral vector Gene Trap Tag http://www.fhcrc.org/labs/soriano/trap.html

  5. Gene Trap Mutagenesis Plasmid vector Gene Trap Tag

  6. pA Event:Tag • X • 2 X • 3 X • 4 X • 5 X • 6 X • 7 X • 8 X • 9 X • 10 X • 11 X

  7. Alignment of Gene Trap Tags BLAST alignment Pct identity 95% Gene tag pct > 50% (ave. 85%) BLAT alignment Pct identity 95% Best 1% kept Gene tag pct > 10% (ave. 92%) DoTS transcripts Genomic DNA Sequence

  8. Gene Trap Tags are Aligned to DTs and Genomic Sequence

  9. From Sequence Tag Alignment to Phenotype

  10. Gene with Isoform-Specific Gene Trap lines.

  11. Gene Trap Alignment Discovery of Alternatively Spliced mRNA

  12. Atlernatively Spliced mRNA?

  13. Number of Distinct Gene Trap Tags Aligned To Genome

  14. Genomic Alignment of Gene Trap Tags Total Number of Alignments

  15. Chr5:1-76 Mb Genes with Gene Trap Tag Alignments

  16. Genomic Alignment of Gene Trap Tags Total Number of Alignments

  17. Sema3a Dpp6, Shh Bst1 Sod3 Pgm1 Kit Hdh Hotspots of Gene Trap Insertions in Chr5:1-76Mb

  18. Chr5:1-76 Mb Genespotential hot spots for gene trap mutagenesis Known genes: Mus musculus insulin degrading enzyme 295 gene tags chr5:22253390-22313497 Amyloid B A4 Precursor Protein Binding Family B Member FE65 37 gene tags chr5:65325098-65642294 ubiquitin-protein isopeptide ligase (E3)41 gene tags chr5:27838650-27895425 Unknown genes: 100% identity to 100% of (BC013481) Unknown 40 gene tagschr5:65066612-65120691

  19. Why do we care if hot spots exist? • How practical are efforts to saturate the genome with gene trap vector insertions? • Insight into evolutionary genomic processes

  20. Gene Trap Integration“Hot Spots” –Future Plans • Do hot spots exist? • Will hot spots limit the practicality of generating gene trap mutants for most genes? • Are gene size and gene tag number related? • Is gene trap mutagenesis related to repeats? • Are there common hot spot motifs?

  21. Do hot spots exist? • Non-trivial problem • Insertion within introns • Insertion site != gene trap sequence tag • Variation of vector design • Definition of hot spot

  22. FYI: defining gene boundary for the distribution graph • For annotation tracks of interest: • total bases in 1Mb bins • genomic gaps: (estimated) gap length • repeats: repeat length • genes/transcripts: exon bases • other alignment features: alignment blocks • total counts (mid-points) in 1Mb bins • union of gene boundaries in 1Mb bins • e.g. this is 100%, not more or less

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