Monitoring Translocation Activity of Transcription Factors and Nuclear Hormone Receptors Using EFC
Poster May 03, 2006
P.A.Fung, K.Peng, B.L.Bosano, P.Kobel, K.R.Olsen and R.M.Eglen
Enzyme Fragment Complementation (EFC) has been adapted to provide a homogeneous assay format suitable for HTS that can be used to study intracellular translocation events without image acquisition.
Here we demonstrate that EFC can provide data for analysis of protein translocation events in whole cells, with the added benefits of increased throughput, simplified luminescence detection, no antibody requirements and decreased assay costs.
For detection of protein translocation to the nucleus, a series of cell lines have been engineered that have restricted expression of the Enzyme Acceptor (EA) fragment of ß gal to the nuclear compartment in live cells. These cells have been used to generate double-stable clones in which a target of interest is expressed as a fusion protein with the ProLabel ß-gal fragment. We demonstrate the application of this approach to screen compounds specific for the glucocorticoid receptor (GR) and NFAT proteins, and provide comparative data for both complementation and imaged based methods.
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