A High Content Primary Screening Assay Identifying Multiple Protein Targets in a Cell Using Quantum Dots
Poster Feb 21, 2006
Christopher Lupton, Dick Ornberg, Marcel Bruchez and Paul Wylie
The development of multiplexing capabilities and high content readouts reporting individual cellular measurements enables assessment of biological variability on a single cell basis. A multiplexed high content screening (HCS) assay allows additional information to be gained from a single assay. One such example is the ability to determine effects of chemical entities on multiple proteins, tested in the same cell.
This approach can greatly increase screening efficiencies and enhance the amount of information achieved from a particular assay. Mitogen-activated protein kinases (MAPKs) are a major target for drug discovery programmes. Using this pathway, we have previously presented data showing the application of HCS to rapidly determine protein kinase activity.
Here, we have used the unique optical properties of semiconductor quantum dots to achieve in-well multiplexing of distinct proteins, including the MAP kinases MEK and ERK. This approach has been applied to rapid high information screening screening approaches in kinase target discovery.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE