Etchevers:Notebook/Genomics of hNCC/2008/11/27

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 * style="background-color: #EEE"|[[Image:C14.jpg|128px]] Genomics of human neural crest cells
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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Progress reports

 * Spent much of the day organizing the rabbit cornea injections data into a powerpoint presentation to discuss with Steph and Angelique. The upshot is that someone will have to cut a lot more slides and that is really tiresome. They could be thicker, though, if they are not too wrinkly and don't come off (they did not until now). However, more antibodies and rinses might be tough on them.


 * Re-read Asma's draft. Don't know what I can contribute until we start getting better results, but will need to read it again and force myself to write.


 * Working on Puting's figure legends and now on the text paragraphs that go with them.


 * Laurence got criticisms back from Nature Genetics that are good but demanding and disappointing for her, and are worth remembering for myself in the future.

This variant segregates with the rare syndrome in the family -- but this variant was found precisely because it is in a region of absolute linkage and sits on a homozygous segment. (Any variant on that haplotype would co-segregate in the family, and had they sequenced the entire region they certainly would have found others.) [...] In general, I tend towards skepticism of arguments in human genetics based on a single mutation. This is particularly of concern when (a) the region of interest is selected out of the genome because it is homozygous and co-segregating with disease, (b) only a single candidate gene in the region is studied, (c) the "control" group for genotyping and sequencing is not similarly consanguineous and ethnically matched, (d) there isn't a prior body of knowledge that can be used to directly relate functional in vitro assays to the human phenotype.

For (c), the control group was as good as it is going to get, I believe. There are a lot of other interesting genes in the region, but many of them are known in quite different pathologies.

This comment by another reviewer made me a little sad, because the expression section was my contribution, and the clinical signs do correlate with the expression pattern to a great extent:

"The expression section really does not offer much insight. Multisystem malformations can be associated equally with tissue specific and ubiquitously expressed genes. A supplementary figure would be more appropriate here.  Far more interesting would be the subcellular (sub-nuclear?) localisation of the gene product, and any changes of this expression during the cell cycle, and in the presence of the mutation."

Another one of the co-authors cogently replied: "I don’t think we are going to be able to get around the comments of reviewer 2 without another independent family... The other issue is that the mouse KO is likely to appear in Nature soon and does NOT have a polymalformative phenotype will cause more reasons to worry that there is some other gene in the linkage interval causing the problem in that family. [...] I would not be optimistic about doing some cell work and going back to NG..."

Which is a bummer for Laurence and for all of us. But the advice is right, just doing the cell work as per the latter reviewer's suggestion won't suffice. And they have been looking for another family for 18 months now.


 * Go back to Asma's and Puting's papers!


 * Heather 09:55, 27 November 2008 (EST):

Tomorrow, Angelique will apply the following protocol to the following slides:

1281-08 (930) I-OD 15 1281-08 (930) II-OG 20 1283-08 (995) I-OD 20 1284-08 (004) I-OD 20 and put the donkey anti-goat on slide 14 from 1281-08 I-OD.

1.Déparaffiner sections: 3x 5’ xylène (ou toluène ou Histoclear) 2x 2’ 100% EtOH 2’ 95% EtOH 2’ chaque of 70%, 50%, 30% EtOH Passage dans de l’eau Passage dans PBS

2.Glycine (0.15% w/v in PBS) 10 min. 3.Passage PBS. 4.Bloquer d’abord au 10% goat serum in PBS pour 30’. Utiliser 300ul par lame sans lamelle en chambre humide. 5.Mettre deux gouttes Image-it Fx (Invitrogen I36933) pour 30’ sous Parafilm. 6.Rincer au PBS. 7.Diluer anticorps anti-fluo au 1:200 (2 ul) et anti-PGD2 au 1:50 (8 ul) dans 390ul du même 10% sérum/PBS. Par ailleurs, diluer donkey anti-goat-Alexa 488 au 1:100 (1 ul dans 99 ul PBS) et appliquer sur la lame témoin. •100 ul par lame sous lamelle en chambre humide. •2 heures température ambiante. 8.5x PBS pour 5’ chaque fois sauf pour la lame témoin, à laisser. 9.Appliquer 2º goat anti-rabbit Alexa 488 sur les lames expérimentales 1:100. 10.5x PBS pour 5’ chaque fois pour toutes les lames. 11.Egoutter jusqu’à près d’être sec, monter en ProLong Gold d’Invitrogen/Molecular Probes avec DAPI (P-36931): chauffer a température ambiante avant utilisation mais garder sous dessication à -20ºC sinon. 12.Laisser prendre 24h à 4ºC avant de mettre du vernis autour.

I also put the coverslips on the anti-tyrosinase, anti-CNPase double stained cell slides. Make sure to feed cells tomorrow.
 * Heather 11:31, 27 November 2008 (EST):


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