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Transient overexpression or tagged cell lines?


The workhorse of cell regulation studies has been the transient overexpression of proteins of interest. Massive overproduction of a protein runs the risk that it enters incorrect cell compartments and now interacts with proteins that it never normally contacts. Thus, inferring cellular location is risky, while overexpression should never be used to provide material for proteomics studies.


Fortunately, advanced recombineering technology now allows stable cell lines to be produced that express tagged proteins at approximately natural levels. These tools can give reproducible results across different labs with methods like RNAi and proteomics, can be studied through the cell cycle, and (for tagged stem cell lines) during and after differentiation. The following table contrasts artefact-prone overexpression with engineered cell lines for various experimental considerations.


Features of Cell Regulation / Effect on Experiment Over Expression Native Expression
Low molecule number X
Spatially arranged protein X
Coupled mRNA transport / Spatial translation Overload system
Mutants that are (unknowingly) unfolded Amyloid ?
Balanced gene dosage of regulators X
Kinases and their substrates are scaffolded X
Laser bleaching to study diffusion of signalling protein Meaningless X/√
Protein complex by Co-IP ???
Proteomics X
Reproducibility ?


For these reasons and more, the partners of the DiGtoP consortium are working to promote and apply a range of recombineered cell lines and engineered mice to study important disease proteins. We will pioneer methods to rapidly produce new cell lines for use in the new era of cell signalling studies. We foresee a time when transient transfections can be relegated to a role in quick and dirty initial studies that will then be carefully followed up with the appropriately recombineered tools.