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Issue 1, 2012
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Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters

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Abstract

We sought to characterize and compare wild-type and oncogenic Ras over-expression. Because different levels of Ras over-expression can have different effects on cell phenotype, it was important to evaluate a wide range of expression. Different expression levels were achieved by using retroviral vectors equipped with different strength promoters. Cells were “shotgun” transduced with a mixture of these vectors to generate heterogeneous populations exhibiting a range of expression levels. We used flow cytometry to analyze the populations and generate high-resolution, nearly continuous Ras dose-response curves. These efforts revealed that a single-copy level of oncogenic Ras generated maximal imatinib resistance and activated MAPK pathway signaling as effectively as six-fold amplification of wild-type Ras. Although further increased expression lead to even greater signal transduction, this increased expression had minimal or decreasing effects on the proliferation rate. In addition, this study introduces a general method to quantify genetic dose-response relationships and identify gene expression ranges that produce an optimized phenotypic response.

Graphical abstract: Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters

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Supplementary files

Article information


Submitted
11 Aug 2011
Accepted
04 Nov 2011
First published
23 Nov 2011

Integr. Biol., 2012,4, 108-114
Article type
Technical Innovation

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