Biomedical Engineering Reference
In-Depth Information
animals, testing transgenic individuals, preparing lymph nodes for primary cell
isolation, and allowing cells and vesicles to interact in the presence of compounds
and staining agents. Due to the length of the sample preparation, well contents were
fixed for analysis on a subsequent day. The data were displayed in quadrants with
positive and negative cell populations, each with the potential of binding the vesicles.
Data analysis required not only identifying individual wells but also quantifying
quadrants for each of the 325,000 compound sets.
4.4.14 Chemical Screen of TOR Pathway GFP Fusion Proteins in
S. cerevisiae
(1R03MH086450-01, Cell-Based Multiplex)
TOR proteins (target of rapamycin) are ser/thr protein kinases phylogenetically
conserved from yeast to man. Rapamycin-sensitive TORC1 signaling pathway
promotes protein synthesis and other anabolic processes, while inhibiting macro-
autophagy and other catabolic and stress-response processes. As a central controller
of cell growth, TORC1 has emerged as a target for drug discovery. Recently, many
components or readouts of TORC1 signaling pathway have been identified through
classic genetic or chemical genetic approaches. Through the screening of the whole
GFP-tagged yeast strain collection (4159 strains), M. Werner-Wasburne and C. Allen
identified several yeast strains showing GFP expression change upon rapamycin
treatment. The rapamycin responsive genes are classified as readouts of four branches
of TORC1 signaling pathway mediated by RTG1/RTG3, GLN3, MSN2/MSN4
transcription factors, and YAK kinase. We used these yeast GFP strains to carry out
a 5-plex high-throughput assay. The yeast GFP strains are differentially stained with
AlexaFluor 405 and AlexaFluor 633 and incubated with the compound for 3 h. The
GFP signal is measured by HyperCyt/CyAn high-throughput flow cytometry. The
purpose of this HTS screen is to identify small-molecule modulators of protein targets
in the TORC1 pathway. The screen will detect structurally distinct, but functionally
rapamycin-like compounds as well as the compounds that regulate individual or
multiple branches of TORC1 signaling pathway.
Lessons Learned : Individual yeast strains have been color coded using AlexaFluor
dyes excited at 405 and 633 nm. GFP is excited at 488 nm. In principle, because yeasts
are heterogeneous, two intensities of staining at 405 and 633 each, along with their
single- and double-strained combinations lead to detection of nine populations.
However, in a 3 h exposure to compounds, the populations blur as unstained
daughter cells are produced. We have found that a fiveplex can be followed (405
low, 405 high, 633 low, 633 high, and 405/605 high. Batch staining of cells for
screening each day has turned out to be tedious.
4.4.15 A Yeast HTS for Caloric Restriction Mimetics That Inhibit
Age-Related Superoxide (1R03MH087439-01, Cell-Based Singleplex)
Reactive oxygen species (ROS) and activation of growth signaling pathways are
important components of aging and contribute to a variety of age-related pathologies,
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