User:Karmella Haynes/Notebook/Polycomb project

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Project Description/Abstract

Characterization of the chromatin protein Polycomb (Pc) as a BioBrick module. Pc usually silences genes marked with histone methylation at H3K27. Can a Pc domain recruit VP64 to a target gene and activate it?

Notes

  • Test Gal4-mCherry-VP64 transactivators on luc reporter:
    • Luc alone: success ✓ 11/12/09 (See 11/19/09 for protocol)
    • Pc-repressed luc: pilot success ✓; full set plus controls success ✓ (See 11/29/10)
  • Pc-ATF stable lines
    • Pc-mCherry-VP64 set (8 lines): Dox+ Western blots, success ✓ 2/18/10; Dox +/- Western blots (done)
    • Pc-mCherry without VP64 (6 neg ctrl. lines): transient check, success ✓ 4/22/10; Dox -/+ Western 5/23/10 success ✓
    • All lines: mCherry vs. DAPI microscopy; done ✓
  • Test Pc-mCherry-VP64 fusion proteins for gene activation activity:
    • Nanostring expression profiles: in progress (collab w/ Casey); done
    • RT-PCR: pending Nanostring results success ✓
    • X-linked Chromatin myc ChIP: myc-specific pull-down successful; ChIP mapping success ✓
  • Test neo-inducible Gal4-EED expression:
    • Test RFP control constructs: ambiguous
    • Gal4-EED constitutive vs. neo-inducible: failed
  • Histone methylation sensor cell lines
    • Expression test (transient): success ✓ 4/20/10
    • Stable Flp-in T-REx YFP reporter lines: KAH96, KAH142, 146, 147 in progress; Gal4-ATF induction control failed (rebuild )


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