DiGtoP Projects
Putting the genes into networks
For each chosen gene we will:
- Tag the protein at either N or C terminus, or both, by recombination into the endogenous gene or BAC transgenesis to ensure that expression is physiological from the endogenous regulatory elements.
- Because the main tag includes GFP, the subcellular localization will be documented in living cells throughout the cell cycle using an HT process that is already established.
- Determine the interaction partners by mass spectrometry.
- Direct validation of the mass spec results will be achieved by sequential tagging to navigate the proteome. That is, proteins identified as interactors with the tagged protein become candidates for tagging in the next round to look for reciprocal identifications.
- Rapid functional validation will be sought using genome-wide esiRNA knock-down libraries and methodolgy established, in part, during NGFN2. The read-out for RNAi knock-downs will be loss or altered localization of GFP signals from candidate protein-protein interaction partner and pathways.
- Evaluation of proteomic phenotypes after RNAi knock-down of key proteins. Recent developments permit a top down examination of total protein profiles from a mammalian cell. We will apply this method to define broad networks and pathway interactions.
- Functional analyses of key proteins in cell assays and conditional mouse models.
- Establishment of protocols to differentiate ES cells into pure populations of selected cell types, so that the engineered ES cells can be used in multiple ways, not just for undifferentiated ES cells or NS cells, as proposed above.
- Tool development to expand options for alternative tagging and functional analyses, particularly to develop (i) a generic and ligand regulatable shRNA strategy against the tag; and (ii) options for targeting at N-terminae.