I had a conversation with a colleague about how to study a gene involved in a rare human lethal disease. A set of mutations in this gene favors the apparition of the symptoms and only a tiny number of samples is available . How can we learn more about this gene ? I've just got a one or two ideas (hey ! I've not been working at the bench for 10 years
:-)...)
- using the product of this gene as a bait in a Yeast two hybrid screen to find one or more cellular partners to this protein.
- A Q-PCR (or chip ?) for a few candidate genes to see how they are expressed in different tissues on affected/non-affected
- ?
hum... that's short. Any other idea ?
In the case of Y2H, you would definitely need to do a cellular localization assay (fluorescent markers) to see if the interactors localize to the same organelle/to the cytoplasm. Other interaction assays include TAP-tagging, coupled to mass spectrometry and the like..
ReplyDeleteIf it's a transcription factor you could do something like ChIP-sequencing to see to which DNA sequences it binds.
Synthetic lethality in yeast could identify involvement in certain pathways / with other genes(but then your targetgene does need to have a yeast-ortholog).
And there's nothing wrong with good ol' in silico predictions of course ;)
Thanks Lucas, This protein is not a transcription factor and has no ortholog is yeast. But TAP-tagging would be a good start !
ReplyDeleteWhat do you know about the effect of the disease ? Symptoms, metabolic alterations, physiological effects … These types of information can provide idea about the target of your gene.
ReplyDelete