Protected: project 2: 02/16/15

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Protected: project 2: 02/13/15

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Bo Huang: Seminar 02/12/15

Life inside the Cell: CRISPR GFP and Imagenomics

Background

  • Joined UCSF in 2009. Recently tenured
  • Packard fellow and New innovator award (well deserved)

Intro

  • studying cars. Conserved gene driving.
  • one knockout not distinguish method.
  • physical biology approach — direct observation.
    • can see both camps are right – reduce friction and provide driving force.

Projects overview

  • centrosome
  • GPCR signaling
  • the genome.

Genome imaging

  • 100 bp is 30 nm, routine for super-resolution

Bo: what we want

  • sequence flexibility
    • label all DNA or all histone, hard to interpret
    • e.g. Wu lab, Cremer lab, Bo lab FISH studies
    • not what Bo’s been going after
  • minimal perturbation, live cell

Sequence speicfic live labeling

  • replication blocks with EdU incorporation?
    • replication blocks not robust
    • cell doesn’t like replication blocking
  • LacO/TetO insertions?
  • 4 papers recently came out in 2013/2014 using TALE-GFP on repetitive sequences in fixed cells
  • Qi Weisseman Lim 2013 CRISPRi (inteference — regulator by Activator / repressor targeting).
  • collaboration with GFP imaging. with Elizabeth Blackburn (telomeres), Weissman and Qi.
  • even leaky expression of GFP on tetR3G is on the edge of too much GFP. (even though this is a special low leaky version of Tet-On).
  • always get nucleolose strong signal. For 3 months no little dots aroudn the cell.
  • get 1 cell in an ‘extreme’ condition. Need extreme lvels of virus delivery (of guide RNA) to get signal.
  • try stem extension (natural squence is longer). 4Us in a row maybe bad. Try AU enginered flip.
  • combine all these together get much better signal.
  • brightness of telomere spots vary. Different telomere lengths?
  • can reduce and increase telomere length and observe change in brightness.
  • do colocalize the telomere binding proteins (in fixed cells) with GFP.
  • other genes? Go after genes and introns with tandem repeats. MUNC4 gene.
  • send cells for sequencing, validate that chromosome number has gone trisomy.
  • design 73 guide RNAs into a pool of viruses (total viral titer 2x previous)
  • 36 guide RNAs sufficient, 26 to 16 doesn’t work.
  • all 3 copies tend to stay on same side of cell (don’t really need live imaging to do this)
  • confined diffusion movie.
  • G2 cells 20 pairs of spots
  • have to dial down laser intensity a lot to take movie
  • Supernova tag (SUnTag) from Vale lab – attach repetitive epitope to protein, get 24 GFPs to attach to this.
  • use split-GFP (used in GRASP and super-res CALM).
  • reduce background to increase contrast.
  • can make YFP or CFP mutation on the split GFP11 component. Photo-activatable GFP.
  • tandem GFP-11s on target protein. add GFP 1-10 to increase signal with high contrast.

Questions

  • perturbation?
    • doesn’t block transcription (does in E coli?)
    • doesn’t block replication
    • local unwiding of DNA
    • nucleosome will have to be moved out of the way. Can’t co-occupy
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Thursday 02/12/15

10:00 am – 1:15 am

Little things

  • some minor fixes to attend to on K99 submission (condensing collaborator/mentor letters)
  • Short planning discussion for project 2
  • some computer issues with chrome and virus scan. Rebooted. Re-initialized scan.
  • flies shipped today
  • still no info from XZ on DGRC login information — probably need to try email.
  • ABSTRACT FOR CSHL BIOLOGY OF GENOMES MEETING!

project 2

  • see post

Seminars

  • see notes from Bo’s seminar
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Protected: project 2

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Tuesday 02/10/15

10:00 am – 7:00 pm, 8:00 pm – 9:45 pm

Project 2

Sequential staining

  • BB reports experiment failed last night. Plan troubleshooting discussion today.
  • apparently a filter issue. staining reported to be sub-par as well.
  • I should probably try these stains myself at some point as a control.

Embryo staining

  • embryos on square cover-glass stained with En look great
  • EN staining is decent given age of antibody
  • took several mosaic regions scans. (probably only saved the EN mosaic and not the WGA. Next time lets save both so we can do the overlay as two separate mosaic files)
  • treated 5 min in Triton, washed with 2x SSC
  • treated 5 min in .1 M HCl, washed with 2x SSC
  • prehybed 10 min in 50% formaimde 2x SSC
  • hybed F02 probe (L3 engrailed locus) with P1 (3 uL of 904) S1 (.6 uL) probe
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Protected: project 2: more results for reviewers

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Monday 02/09/14

10:30 am – 4:00 pm, 5:30 pm – 11:15 pm

Project 2

Communication

  • wrote to YS and CS about STORM2 experiments
  • contacted referees with reminders about K99 letters
  • planned sequential staining experiments with BB for tonight

Planning embryo imaging

  • waiting to hear back about STORM2
  • possibly stain an image embryos tomorrow.

To write

  • Biology of Genomes abstract! (do this tomorrow)
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Protected: project 2: more analysis

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Protected: project 2: 02/09/14

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