User:Pakpoom Subsoontorn/Notebook/Genetically Encoded Memory/2008/10/28

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PhiC31

  • Host: Streptomyces lividans
  • Action: tyrosine integrase
  • Structure:

length:: 613 aa crystal structure is unknown.

  • Target sequence: attB:: has two inverted repeated sequence (core-type) flanking an overlap region (TTG). Minimal attB is 34 bps while minimal attP is 39 bp for e.coli intramolecular integration. attP:: has core-type sites and overlap region, without an arm-type site. --See the schematic sequence of attB and attP at (1-f1). See Groth-2000 for experiment for minimal sequence. --See Thyagarajan-2001 for integration of attB and attP above background/pseudointegration site ~attP in human genome (these could be very cool model experiment!)
  • Change specificity:

Mutation/directed evolution could change specificity to pseudo attP site (HpsA, 44% identity)

  • Mechanism:
  • Excision:

Excisionase has not yet been found.

  • mutant/hybrid
    • directed evolution for targeting human's site
  • Non-natural host:
    • Integration works in E.coli, mammalian and in vitro,
    • Mouse embryos Hollis et al 2003
    • Bovine fiboblase--Ma et al 2006
    • Drosophila--Groth et al 2004
    • Aedes aegyptus--Nimmo et al 2006
    • Silk worm--Nakayama et al 2006
    • cassette exchange: Drosphila --Bateman et al 2006, fission yeast--Thomson et al 2001, mouse embryonic stem cell--Belteki et al 2003
    • Xenopus laevis--Allen&Weeks 2005
    • chloroplast transformation--Lutz et al 2004
  • gene therapy example--table1 for ref-1


References

  • 1Calos MP. The PhiC31 Integrase System for Gene Therapy. Current gene Therapy, 2006, 6, 633-645.


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