Detecting the FRET Response of the GeneBLAzer® Cell Line D1 CRE-bla CHO-K1 to Agonists and Antagonists using Microplate Cytometry
Poster Feb 01, 2007
Christopher Lupton, Randy Hoffman, Paul Wylie and Wayne Bowen
The GeneBLAzer® CHO.K1-D1 cell line (Invitrogen) stably expresses both the ß-lactamase gene downstream of the cAMP response element (CRE) and the dopamine D1 receptor. Stimulation of the cells with dopamine D1 receptor agonists, results in transcriptional activation of the ß-lactamase gene through CRE. A FRET-enabled substrate (CCF4-AM) fluoresces green, in the absence of ß- lactamase reporter activity, and blue when cleaved. This technology has been measured by bulk fluorescence readers, which report data on a whole well basis.
In this study, the violet laser in the Acumen Explorer 405 microplate cytometer was used to excite CCF4-AM substrate and the resulting fluorescent emissions simultaneously detected in the blue and green channels.High content data for dopamine D1 receptor activation were calculated from ratios of blue to green fluorescence in cell populations. We have shown that the Acumen Explorer 405 has the ability to accurately measure ß- lactamase activity, and generates good fold activations above baseline.
We have also shown that we can obtain toxicological data from the same plate, thereby providing additional, valuable information during the screening process. When coupled with short read times of less than 10 minutes per plate, this technology provides an excellent opportunity for functional genomic applications.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE