Determining subcellular localization of novel drug targets by transient transfection in COS cells |
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Authors: | Iris Simon Meredith Wright Tanya Flohr Peter Hevezi Ingrid W Caras |
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Institution: | (1) Eos Biotechnology Inc., 225A Gateway Blvd., South San Francisco, CA 94080, USA;(2) Eos Biotechnology Inc., 225A Gateway Blvd., South San Francisco, CA 94080, USA |
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Abstract: | Genomics-based approaches are increasingly being used to identify disease-associated genes that represent potential new drug
targets. As a first step in the validation of genes of unknown function, we describe a method for rapidly determining the
subcellular localization of the gene product. If an immunotherapeutic approach is being considered, it is of particular interest
to identify targets that are either on the cell-surface or secreted. Transient expression in COS cells combined with immunofluorescent
staining provides a semi-high throughput method for determining the subcellular localization of multiple targets in parallel.
COS cells are ideal for this purpose since: (i) they transfect easily; (ii) the high levels of expression that can be achieved
transiently allow detection after 24 h; and (iii) the relatively large size and spread morphology of these cells allows the
subcellular organelles to be easily visualized. To evaluate the system, we show prototype staining patterns for known cytoplasmic,secreted,
Golgi-associated, endoplasmic reticulum-associated, and plasma membrane proteins, as well as data for novel targets. The localization
of novel secretory and cell-surface proteins as determined by immunofluorescent staining, was confirmed by independent methods.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | immunofluorescent staining surface biotinylation transient transfection |
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