Etchevers:Notebook/Genomics of hNCC/2008/08/04

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What's going down with hNCC and in situ hybridizations in early August

Wrote reply to editor for HMG draft on NCC stem cells.

Sophie has a number of projects underway:

  • With Jeanne, who wants to know whether or not SNAI2 directly binds the promoter of RET, eventually within the first intron, I suppose? Ideally within enteric ganglia. Had problems with getting hNCC to grow, even when they did, the ChIP gave ok + control but no signal for amplicon of SNAI2 on RET promoter even after 40 PCR cycles – but what was chosen as the promoter? Did they do genomic amplification to have lots of immunoprecipitate to work with?
  • Cells grow well on commercial but not manually coated Labtek slides. This was necessary for immunohistochemistry for cilia and beta-tubulin. Sophie says saw some wonderful mitoses. Now there are cilia on there, then what? (Something with Sophie Saunier and Tania).
  • Millipore had sent inserts because Sophie is interested in migration and invasivity. She manually coats the membranes with collagen I. It penetrates into the surface to some extent, yet somehow growth factors will not cross the insert. The idea is to see what can cross and if you can promote or prevent it with media treatments. Added more serum underneath relative to top so as to promote migration. I don’t know if that is necessary or even desirable.
  • Karyotypes relaunched at passage 30 for (which?) lines.
  • We need to find out about the right to distribute our cells. I must write back the Japanese fellow who contacted us, as well as write the stupid dossier(s?) for the Agence de Biomedecine.

Candice is undertaking a certain number of ISH.

  • The earliest one was on the C20 embryo she had sectioned, but did not work for LHX3/4 and not even very well for ISL1. Then again the + control did not work either.
  • Second HIS was with Emmanuelle for the candidate gene she discussed the other week) and that didn’t work either. Lots of blue background and not much signal. I hypothesized that under the current lab conditions of extreme heat, the pH of the Tris 9.5 when diluted to working 100mM (I think) might be too low for the NBT-BCIP step. Candice assured me that she is using fresh levamisole so that’s not it. It was 32°C in the lab when we spoke; sometimes it’s 35º in the summer, as opposed to the optimal 20-22º.
  • However, I took a look and the control (sense) slides are perfectly white. So it’s not a pH thing, and it’s not so uniformly blue as that. Bubble perhaps on the positive control slide (which definitely did not work – ISL1 near sections that had previously worked well)? Need to redo, as I think that there is specific signal in some areas. Can then verify certain interesting structures eg. the developing cerebellum?
  • Have not yet looked at LHX3/4.
  • Manuscript from Laurence Colleaux to add to: scale bar, legend and paragraph. Finish tomorrow.

I also set up requests for reimbursement to Institut Necker - need to find reference for ISSCR expenses, make new "bon de commande" (purchase order) for Easyjet to SciBlog 2008 conference - don't forget housing expenses, later - and today's flight and Air France transport.

  • Alethea 16:58, 4 August 2008 (UTC):


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